Method for treatment and control of plant disease

ABSTRACT

The present invention provides methods for development of a virulent bacteriophage-based treatment for the control of plant diseases caused by  Xylella fastidiosa . The invention further provides methods of isolating and propagating bacteriophage virulent to  X. fastidiosa  in a  Xanthomonas  bacterial host and for treating or reducing symptoms of  X. fastidiosa  infection in a plant. The invention further provides methods of isolating and propagating bacteriophage virulent to  Xanthomonas axonopodis  pv.  citri  and for treating or reducing symptoms of  Xanthomonas axonopodis  pv.  citri  infection in a plant.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. provisional applications No.61/716,245, filed Oct. 19, 2012, and No. 61/785,535, filed Mar. 14,2013, herein incorporated by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

The U.S. government has certain rights in this invention, pursuant tothe following: Animal & Plant Health Inspection Service (APHIS)Cooperative Agreement Award for the Texas Pierce's Disease Research &Education Program, Agreement Number 11-8500-0955-CA, with AgriLifeResearch; and Otsuka Pharmaceutical Co., LTD, Agreement number 406039,with AgriLife Research.

INCORPORATION OF SEQUENCE LISTING

The sequence listing that is contained in the file named“TAMC019US_ST25.txt,” which is 907 kilobytes as measured in MicrosoftWindows operating system and was created on Oct. 17, 2013, is filedelectronically herewith and incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to the field of plant pathology. Morespecifically, the invention relates to methods and compositions forisolating bacteriophage and for treatment of plant diseases caused byXylella fastidiosa and Xanthomonas axonopodis comprising use of abacteriophage, a virus of bacteria.

BACKGROUND OF THE INVENTION

Bacteria can cause many diseases in plants, including Pierce's Diseaseof grapevines, and Citrus Canker of citrus plants. The bacteria infectplant tissues and can cause wilting, poor growth, lesions on fruit, andeven plant death. Infection can occur through spreading by wind, rain,contaminated equipment, or vector insects, rapidly spreading to otherplants, and resulting in deleterious effects to the plant and massivecrop losses. Effective treatment of these diseases requires a method oftreating the plant to eliminate the bacteria.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a method of propagating avirulent bacteriophage (phage) that includes X. fastidiosa in its hostrange, comprising infecting a culture of Xanthomonas bacteria with thebacteriophage, allowing the bacteriophage to propagate, and isolatingbacteriophage particles from the culture. In another embodiment, theXanthomonas bacteria comprises species strain EC-12. In anotherembodiment, the bacteriophage infects the cell by binding to a cellsurface feature. In another embodiment, the cell surface feature is aType IV pilus. In another embodiment, the bacteriophage comprises atailed bacteriophage from the group consisting of a podophage, asiphophage, and a myophage. In other embodiments, the bacteriophage isisolated from the environment, a sewage treatment plant, or effluent, aplant, or a surface thereof or from the surrounding soil. In otherembodiments of the present invention, a surrogate host is used to enrichfor virulent bacteriophage. In still another embodiment, thebacteriophage is virulent in Xylella fastidiosa. In other embodiments,agar overlaying is used for growth of the bacteriophage.

In another aspect, the invention provides a method of obtaining acandidate biocontrol agent for Pierce's Disease comprising contacting X.fastidiosa and Xanthomonas bacteria with a sample comprising apopulation of virulent bacteriophage and isolating at least a firstbacteriophage from the population capable of lysing said X. fastidiosaand Xanthomonas bacteria. In one embodiment, the bacteriophage infects acell by binding to a cell surface feature. In another embodiment, thecell surface feature is a Type IV pilus. In still another embodiment,the cell surface feature is required for pathogenesis/virulence of thebacterial host. Other embodiments include contacting a lawn of at leastone of X. fastidiosa and Xanthomonas with the sample, contacting the X.fastidiosa and Xanthomonas with the sample simultaneously, andcontacting the X. fastidiosa and Xanthomonas with the samplesequentially. In other embodiments, the bacteriophage is isolated fromthe environment, a sewage treatment plant, or effluent, a plant, or asurface thereof or from the surrounding soil. In another embodiment, thebacteriophage used is virulent in Xylella fastidiosa. The method mayfurther comprise detecting lysed bacterial host cells, or plaqueformation, after contacting host bacteria with the virulentbacteriophage. In particular embodiments, the method comprises a plateagar overlay or a plate of the bacterial host cells onto which a sampleof bacteriophage have been introduced.

In other embodiments, the bacteriophage is prepared by use of a softagar overlay containing the X. fastidiosa and Xanthomonas, and infurther embodiments, high-titer phage plate lysates are prepared byharvesting one or more overlay plate(s) comprising a X. fastidiosastrain or a Xanthomonas strain, such as EC-12, exhibiting confluentlysis, followed by maceration and clarification by centrifugation. Afterbeing filter sterilized, the resulting lysates may be stored, forinstance at 4° C. Subsequently, high-titer phage lysates are purified,for instance by isopycnic CsCl centrifugation, and extracted phagesolution are dialyzed. The resulting CsCl-purified bacteriophagetypically displays a titer of about 1×10¹¹ PFU/ml.

In some embodiments, a ratio of bacteriophage in plant tissue filtrates(PTFs) is about 1 ml of PTF to 20 ml the surrogate host (activelygrowing culture of selected host) for 4 days for X. fastidiosa strainTemecula or for 4 h for Xanthomonas strain EC-12.

Another aspect of the invention provides a method of preventing orreducing symptoms or disease associated with X. fastidiosa in a plant,comprising contacting a plant with bacteriophage that includes X.fastidiosa in its host range, wherein the symptoms or disease associatedwith X. fastidiosa comprise typical Pierce's Disease (PD) symptoms,wherein the leaves display a yellow or red appearance along margins,with eventual leaf margin necrosis. In one embodiment, the bacteriophageparticles may be introduced into the plant. In another embodiment, theplant is selected from the group consisting of a grapevine plant, acitrus plant, almond, coffee, alfalfa, oleander, oak, sweetgum, redbud,elm, peach, apricot, plum, blackberry, mulberry, and Chitalpatashkentensis. In another embodiment, the bacteriophages are introducedinto the plant by injection, an insect vector or delivered via the rootsystem by injection. In other embodiments, injection comprises a needleor a needle-free system, a pneumatic air or pressure injection system.In other embodiments, the injection is performed manually, or once, ormore than once. In another embodiment, the insect vector is a glassywinged sharpshooter. In another embodiment, the bacteriophage to beintroduced into the plant is from 1 to 10¹² PFU/ml (plaque formingunits/ml), 10⁴ to 10¹¹ PFU/ml, and 10⁷ to 10¹⁰ PFU/ml. In anotherembodiment, the bacteriophage particles are obtained by a methodcomprising infecting a culture of Xanthomonas bacteria with thebacteriophage, allowing the bacteriophage to propagate, and isolatingbacteriophage particles from the culture. In another embodiment, themethod comprises contacting a population of plants with thebacteriophage particles to prevent or reduce symptoms associated with X.fastidiosa. In still another embodiment, the bacteriophage comprises atleast one bacteriophage (phage) of a strain selected from the Xfas100phage type or the Xfas300 phage type, described below.

In another aspect, the invention provides a plant disease biocontrolcomposition formulated for delivery to a plant, the compositioncomprising at least one diluent, adjuvant or surfactant, and at leastone bacteriophage from the Xfas100 phage type or the Xfas300 phage type,described below. In one embodiment, the composition is further definedas being formulated for introduction to a plant via injection, spraying,misting, or dusting. In another embodiment, the composition is furtherdefined as being formulated for topical administration to a plant.

In another aspect, the invention provides a method of obtaining acandidate biocontrol agent for citrus canker comprising contactingXanthomonas axonopodis pv. citri bacteria with a sample comprising apopulation of virulent bacteriophage and isolating at least a firstbacteriophage from the population capable of lysing said Xanthomonasaxonopodis bacteria. In one embodiment, the bacteriophage infects a cellby binding to a cell surface feature. In another embodiment, the cellsurface feature is a type IV pilus. In still another embodiment, thecell surface feature is required for pathogenesis/virulence of thebacterial host. Other embodiments include contacting a lawn ofXanthomonas with the sample. In another embodiment, the bacteriophageused is virulent in Xanthomonas axonopodis.

Another aspect of the invention provides a method of preventing orreducing symptoms or disease associated with Xanthomonas axonopodis in aplant, comprising contacting a plant with bacteriophage that includesXanthomonas axonopodis in its host range. In one embodiment, thebacteriophage particles may be introduced into the plant. In someembodiments, the plant is a citrus plant selected from the groupconsisting of a Citrus spp., a Fortunella spp., a Poncirus spp., a lime,a lemon, an orange, a grapefruit, a pomelo, and hybrids of trifoliateorange used for rootstocks. In another embodiment, the bacteriophagesare introduced into the plant by injection, by an insect vector, or isdelivered via the root system by injection. In some embodiments,injection comprises a needle or a needle-free system, a pneumatic air orpressure injection system. In other embodiments, the injection isperformed manually, or once, or more than once. In another embodiment,the insect vector is a glassy winged sharpshooter. In anotherembodiment, the bacteriophage to be introduced into the plant is at aconcentration of from 1 to 10¹² PFU/ml (plaque forming units/ml), 10⁴ to10¹¹ PFU/ml, and 10⁷ to 10¹⁰ PFU/ml. In another embodiment, the methodcomprises contacting a population of plants with the bacteriophageparticles to prevent or reduce symptoms associated with Xanthomonasaxonopodis and pathovars thereof in the population. In still anotherembodiment, the bacteriophage comprises at least one bacteriophage of astrain selected from the Xfas100 phage type or the Xfas300 phage type,described below.

In another aspect, the invention provides an isolated bacteriophage thatis virulent to Xanthomonas axonopodis a Xfas303 bacteriophage, wherein arepresentative sample of said bacteriophage has been deposited underATCC Accession Number PTA-13099. In yet another aspect, the inventionprovides an isolated bacteriophage that is virulent to Xanthomonasaxonopodis and/or X. fastidiosa as one of bacteriophage selected fromthe group consisting of: Xfas101, Xfas102, Xfas103, Xfas104, Xfas105,Xfas106, Xfas107, Xfas108, Xfas109, Xfas110, Xfas301, Xfas302, Xfas304,Xfas305, and Xfas306, wherein representative samples of saidbacteriophage Xfas103, Xfas106, Xfas302, Xfas303, Xfas304, and Xfas306have been deposited under ATCC Accession Number PTA-13095, PTA-13096,PTA-13097, PTA-13098, PTA-13099, and PTA-13100.

In certain embodiments, the invention provides a method of preventing orreducing symptoms or disease associated or caused by X. fastidiosa orXanthomonas axoxonopodis pv. citri in a plant comprising a step ofcontacting said plant with a virulent bacteriophage which includes X.fastidiosa and/or Xanthomonas axoxonopodis pv. citri in its host range,further wherein the bacteriophage is at least one bacteriophage selectedfrom the group consisting of the Xfas100 phage type, and the Xfas300phage type, wherein the Xfas100 type phage has at least onecharacteristic selected from the group consisting of (a) thebacteriophage is capable of lysing said Xylella fastidiosa and/orXanthomonas bacteria; (b) the bacteriophage infects a cell by binding toa Type IV pili; (c) the phage belongs to a group of tailed bacteriophageexhibiting long non-contractile tails with capsid ranging from 55-77 mmin diameter, a morphology typical of Siphoviridae family; (d) thegenomic size of bacteriophage is about 55500 bp to 56200 bp; and (e) thebacteriophage prevents or reduces symptoms associated with Pierce'sDisease in a plant or plants; and wherein the Xfas300 type phage has atleast one characteristic selected from the group consisting of: (a) thebacteriophage is capable of lysing said Xylella fastidiosa and/orXanthomonas bacteria; (b) the bacteriophage infects a cell by binding toa Type IV pilus; (c) the phage belongs to a group of tailedbacteriophage exhibiting short non-contractile tails with capsid rangingfrom 58-68 mm in diameter, a morphology typical of Podoviridae family;(d) the genomic size of bacteriophage is about 43300 bp to 44600 bp; and(e) the bacteriophage has an activity of preventing or reducing symptomsassociated with Pierce's Disease in a plant or plants. In certainembodiments, a single type of virulent bacteriophage is introduced intoa plant; in other embodiments, a combination of 2, 3, 4, 5, 6, or morevirulent bacteriophage isolates or types are introduced into a plant,either simultaneously or sequentially. In certain embodiments, thebacteriophage comprise a genome with a DNA sequence selected from thegroup consisting of SEQ ID NO:11-24, or a DNA sequence at least 90%,95%, 98%, or 99% identical thereto. Thus, in certain embodiments, thebacteriophage to be introduced into a plant is selected from the groupconsisting of: Xfas101, Xfas102, Xfas103, Xfas104, Xfas105, Xfas106,Xfas107, Xfas110, Xfas301, Xfas302, Xfas303, Xfas304, Xfas305, andXfas306. Plant disease biocontrol compositions formulated for deliveryto a plant, and comprising such Xfas100 and/or Xfas300 typebacteriophage are also contemplated. The biocontrol composition mayfurther comprise a carrier. In some embodiments the carrier may comprisea diluent, a surfactant, and/or a buffer.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the features and advantages of thepresent invention, reference is now made to the detailed description ofthe invention along with the accompanying figures and in which:

FIG. 1: Shows a TEM image of phages Xfas302, Xfas303, Xfas304, andXfas305, with morphology and size characteristic of Podoviridae.

FIG. 2: Shows a TEM image of phages Xfas101, Xfas102, Xfas103, andXfas104, with morphology and size characteristic of Siphoviridae.

FIG. 3: Shows Podoviridae and Siphoviridae bacteriophages of X.fastidiosa isolated from wastewater, able to form plaques on XF15 andEC-12.

FIG. 4: Shows a genomic map of Siphoviridae Xfas103 and Xfas106.

FIG. 5: Shows a genomic map of Podoviridae Xfas302, Xfas303, Xfas304,and Xfas306.

FIG. 6: Shows a grapevine plant exhibiting symptoms of Pierce's Disease8 weeks after inoculation with strain XF54 and not challenged withbacteriophage.

FIG. 7: Shows a summary of the grapevine bacteriophage therapeutic andpreventative challenge study.

FIG. 8: Shows movement and persistence of individual bacteriophages ininoculated grapevines at 8 (Top) and 12 (Bottom) weeks after phageinoculation. Left panel: phages present in root tissue. Middle panel:phages present in cordon 1 of grapevine. Right panel: phages present incordon 2 of grapevine.

FIG. 9: Shows levels of XF15 in inoculated grapevines challenged withphage cocktail 3 weeks later. Samples were collected 9 weeks after phagecocktail challenge (12 weeks after bacterial inoculation). Left panel:bacteria present in root tissue. Middle panel: bacteria present incordon 1 of grapevine. Right panel: bacteria present in cordon 2 ofgrapevine. Gray bars show XF15 levels in XF15 inoculated vines. Blackbars show XF15 levels in XF15 inoculated vines challenged with phagecocktail at week 3-post pathogen inoculation. Arrows show segment withpoint of inoculation. Each bar is representative of average CFU/gpt(gram plant tissue) of roots and 2 cordons for 3 vines.

FIG. 10: Shows levels of cocktail phages in grapevines initiallyinoculated with XF15 and challenged with phage cocktail 3 weeks later.Samples were collected 5, 7, and 9 weeks after phage cocktail challenge(8, 10, and 12 weeks after initial bacterial inoculation). Left panel:phages present in root tissue. Middle panel: phages present in cordon 1of grapevine. Right panel: phages present in cordon 2 of grapevine.Black bar show phage levels in cocktail inoculated plants. Gray bar showphage levels in XF15 inoculated vines challenged with phage cocktail atweek 3-post pathogen inoculation. Arrows show segment with point ofinoculation. Each bar is representative of the average PFU/gpt (gramplant tissue) of 4 phages in cocktail determined from roots and 2cordons for 3 vines.

FIG. 11: Shows levels of phages in grapevines initially inoculated withphage cocktail and challenged 3 weeks later with XF15. Samples werecollected 5, 7, and 9 weeks after XF15 challenge (8, 10, 12 weeks afterinitial phage inoculation). Left panel: phages present in root tissue.Middle panel: phages present in cordon 1 of grapevine. Right panel:phages present in cordon 2 of grapevine. Black bars show phage levels incocktail inoculated vines. Gray bars show phage levels in cocktailinoculated vines challenged with XF15 at week 3-post phage inoculation.Arrows show segment with point of inoculation. Each bar isrepresentative of the average PFU/gpt (gram plant tissue) of 4 phages incocktail determined from roots and 2 cordons for 3 vines.

FIG. 12: Shows results of spot titration of phage Xfas303 on Xanthomonasaxonopodis pv. citri strains.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO:1—The X. fastidiosa-specific oligonucleotide forward primerdesigned for X. fastidiosa gyrB.

SEQ ID NO:2—The X. fastidiosa-specific oligonucleotide reverse primerdesigned for X. fastidiosa gyrB.

SEQ ID NO:3—The bacteriophage Xfas304-specific oligonucleotide forwardprimer designed for the bacteriophage DNA primase gene.

SEQ ID NO:4—The bacteriophage Xfas304-specific oligonucleotide reverseprimer designed for the bacteriophage DNA primase gene.

SEQ ID NO:5—The bacteriophage Xfas303-specific oligonucleotide forwardprimer designed for the bacteriophage DNA primase gene.

SEQ ID NO:6—The bacteriophage Xfas303-specific oligonucleotide reverseprimer designed for the bacteriophage DNA primase gene.

SEQ ID NO:7—The bacteriophage Xfas103-specific oligonucleotide forwardprimer designed for the bacteriophage DNA helicase gene.

SEQ ID NO:8—The bacteriophage Xfas103-specific oligonucleotide reverseprimer designed for the bacteriophage DNA helicase gene.

SEQ ID NO:9—The bacteriophage Xfas106-specific oligonucleotide forwardprimer designed for the bacteriophage DNA helicase gene.

SEQ ID NO:10—The bacteriophage Xfas106-specific oligonucleotide reverseprimer designed for the bacteriophage DNA helicase gene.

SEQ ID NO:11—The genomic sequence of bacteriophage Xfas101.

SEQ ID NO:12—The genomic sequence of bacteriophage Xfas102.

SEQ ID NO:13—The genomic sequence of bacteriophage Xfas103.

SEQ ID NO:14—The genomic sequence of bacteriophage Xfas104.

SEQ ID NO:15—The genomic sequence of bacteriophage Xfas105.

SEQ ID NO:16—The genomic sequence of bacteriophage Xfas106.

SEQ ID NO:17—The genomic sequence of bacteriophage Xfas107.

SEQ ID NO:18—The genomic sequence of bacteriophage Xfas110.

SEQ ID NO:19—The genomic sequence of bacteriophage Xfas301.

SEQ ID NO:20—The genomic sequence of bacteriophage Xfas302.

SEQ ID NO:21—The genomic sequence of bacteriophage Xfas303.

SEQ ID NO:22—The genomic sequence of bacteriophage Xfas304.

SEQ ID NO:23—The genomic sequence of bacteriophage Xfas305.

SEQ ID NO:24—The genomic sequence of bacteriophage Xfas306.

DETAILED DESCRIPTION OF THE INVENTION

The following definitions and methods are provided to better define thepresent invention and to guide those of ordinary skill in the art in thepractice of the present invention. Unless otherwise noted, terms are tobe understood according to conventional usage by those of ordinary skillin the relevant art.

The invention provides, for the first time, methods allowing efficientpropagation and isolation of bacteriophage (phage) capable of infecting,replicating within, and lysing X. fastidiosa and/or Xanthomonasaxonopodis (Xa) and pathovars thereof. The invention also provides amethod for controlling bacterial disease in plants. Plant diseases thatmay be controlled in accordance with the present invention may include,but are not limited to, Pierce's Disease and citrus canker. Bacterialspecies useful in accordance with the invention may include, but are notlimited, to a Xylella species, such as Xylella fastidiosa, or aXanthomonas species, such as Xanthomonas axonopodis and pathovarsthereof, such as Xanthomonas axonopodis pv. citri (Xac).

As used herein, a “bacteriophage” or “phage” refer to a virus ofbacteria. As used herein, “Xanthomonas axonopodis” or “Xa” refers to aXanthomonas axonopodis bacterial species or pathovar thereof, which mayinclude Xanthomonas axonopodis pv. citri (Xac) or any other pathovar ofXanthomonas axonopodis. Currently, propagation of bacteriophage capableof lysing X. fastidiosa is labor-intensive in the laboratory, using X.fastidiosa host cells and complex, expensive media in a solid format.This may require 7-10 days to yield low quantities of bacteriophage. Thepresent invention thus represents a significant advance, providing forpropagation of bacteriophage capable of infecting X. fastidiosa bygrowing the bacteriophage in a fast-growing host bacteria such asXanthomonas species EC-12 to rapidly produce bacteriophage; this isdesignated as the “surrogate host” approach. The technique is fast andcost-effective, capable of use with conventional media componentsavailable in the art. The technique is also amenable to scale-up. Theability to produce virulent phages that lyse (kill) X. fastidiosa and/orXa in a surrogate host that can replicate hourly under standardconditions, instead of days using a host that at best replicates dailyin a very complex media makes viable for the first time the productionand implementation of X. fastidiosa- and/or Xanthomonasaxonopodis-mediated disease control and treatment methods comprising useof virulent phages. Culture of Xa can be performed in nutrient brothwith a generation time of approximately 2-3 hours. However, Xa is apermitted pathogen and thus requires a biosafety level of 2 (BL2) toculture. Therefore, similar to X. fastidiosa, Xa may not be practicalfor large-scale production.

Bacteriophage may be isolated by a soft agar overlay method, allowingfor isolation of phage from X. fastidiosa and/or Xanthomonas cells, andin further embodiments, high-titer phage plate lysates are prepared byharvesting one or more overlay plate(s) of a X. fastidiosa strain or aXanthomonas strain, such as strain EC-12, exhibiting confluent lysis,followed by maceration, clarification by centrifugation, and filtersterilization. The resulting lysates may be stored at 4° C.Subsequently, high-titer phage lysates may be purified for instance byisopycnic CsCl centrifugation, and extracted phage solution can bedialyzed. Resulting CsCl-purified bacteriophage having a titer of about1×10¹¹ PFU/ml can thus be obtained. In other embodiments, bacteriophagein plant tissue filtrates (PTFs) may be filtered. A preferred ratio forfiltration is 1 ml of PTF to 20 ml of the surrogate host culture (anactively growing culture of a selected host), grown, for instance, for 4days for X. fastidiosa strain Temecula or for 4 h for Xanthomonas strainEC-12.

Using methods for the detection and propagation of bacteriophagevirulent to X. fastidiosa and/or Xanthomonas axonopodis (“Xa”)pathovars, virulent bacteriophage which are capable of causing lysis ofX. fastidiosa and/or Xa can be selected from a desired source, such asfrom the environment, including plants, wastewater, and/or soil water,and propagated according to the invention. Bacteriophages that may beidentified in accordance with the present invention may be defined byparticular characteristics as described by Casjens et al. (Research inMicrobiology, 159:340-348, 2008), such as capsid shape and size, genomesize, arrangement of genes and/or gene modules, morphology, and lifecycle. In one embodiment, bacteriophages of the present invention may bevirulent, isometric, with a triangulation number of T=7, a genome sizeof about 60 kb or within about 15% of 60 kb, may include direct terminalrepeats in the genome. The virulent bacteriophage can be used, forexample, to control and prevent disease cause by Xylella species andsubspecies and/or Xanthomonas species such as Xanthomonas axonopodis andpathovars thereof, such as citri.

Currently, five subspecies of Xylella are recognized as causing plantdisease. Plant species able to be infected by Xylella are listed, forexample, at www.cnr.berkeley.edu/xylella/control/hosts.htm, as describedin Hernandez-Martinez et al., (American Phytopathological Society,97(7):857-864, 2007) and Nunney et al., (PLoS ONE, 5(11):e15488, 2010),and may include commercial crops such as, but not limited to,grapevines, citrus, coffee, almond, peach, alfalfa, apricot, plum,blackberry, mulberry, and horticultural plants such as oleander, oak,sweetgum, redbud, elm, and Chitalpa tashkentensis. In one embodiment ofthe invention, bacteriophage can be isolated from environments where X.fastidiosa is unable to grow because of its unique growth requirements.Further, in accordance with the present invention, plant species able tobe infected by a Xanthomonas axonopodis pathovar may include, but arenot limited to, a Citrus spp., a Fortunella spp., a Poncirus spp., alime, a lemon, an orange, a grapefruit, a pomelo, and hybrids oftrifoliate orange used for rootstocks.

The invention thus provides methods for development ofbacteriophage-based treatments for the control of plant diseases causedby X. fastidiosa, which is a xylem-limited, insect vectored,Gram-negative bacterium that causes disease in many plants. Mostnotably, X. fastidiosa is the causal agent of Pierce's Disease (PD) ofgrapes, which is currently a limiting factor in the cultivation of highquality wine grapes in areas of the U.S., including Texas andCalifornia. One important plant disease caused by X. fastidiosa isPierce's Disease (“PD”) of grape, which causes visible symptomsincluding yellowed leaves, or leaves with red along margins. Eventuallydrying and necrosis of leaf margins and leaves may occur. Insect vectorssuch as the leafhopper Glassy Winged Sharpshooter (“GWSS”) may spreadthe disease, as well as phage which infect the disease-causing bacteriaand which may be useful for biocontrol efficacy.

Presently, there are no effective control measures for PD short ofaggressive culling of the infected vines. The current invention permitstreatment of such diseases by providing, for the first time, a viablesystem for generating sufficient bacteriophage quantities in acost-effective manner to permit plant treatments. The invention alsoprovides methods for development of bacteriophage-based treatments forthe control of plant diseases caused by Xa, including Xac, which is thecausal agent of citrus canker. In a particular embodiment, the inventionprovides a method for controlling disease of Xa in a plant.

As used herein, the term “virulent” refers to a virus, particularly abacteriophage, that is able to infect, replicate within, and lyse (kill)a host cell. The term “temperate” refers to a bacteriophage that canintegrate into the host genome (lysogenize) or lyse the host cell. Inone embodiment of the invention, phages are propagated in a suitablehost, as is described herein. The term “host” refers to a bacterial cellthat can be used to produce large quantities of bacteriophage. One stepin the development of a bacteriophage-based control strategy providedherein is the identification and propagation of virulent phages thatrecognize particular bacterial receptor sites. Production and deliveryof bacteriophage virulent to disease-causing bacteria must be economicalto represent a viable biocontrol option.

Phages infect a host cell via recognition of receptors, which caninclude, but are not limited to, surface proteins such as Omp A andOmpF, the core and O-chain of the bacterial LPS in Gram-negativebacteria, sex and type IV pili (e.g. Roine et al., Mol. Plant MicrobeInteract., 11:1048-1056 (1998)), and flagella. Without being limited toany given theory, it is believed that bacteriophage may infect X.fastidiosa and Xa cells via type IV pili. Thus, in one embodiment, ahost according to the present disclosure may be any type of bacteria,and particularly any bacterial species that a virulent temperatebacteriophage, or a derivative thereof, such as a passaged phage, isable to adsorb to and infect via a surface receptor that is required forvirulence and/or pathogenicity, such as a type IV pili or a TonB-likeprotein. By “passaged phage” is meant a phage population which has beenpropagated by one or more periods of growth in cultured host cells.Typical hosts used in the present invention may be bacterial cells,particularly bacterial species of the family Xanthomonadaceae, whichincludes both Xylella and Xanthomonas. In some embodiments, strains ofX. fastidiosa which may be useful in practicing this invention mayinclude Temecula1 (ATCC 700964); Ann-1 (ATCC 700598); Dixon (ATCC700965); XF53, XF54, and XF95 (Whitehorn et al., Science, 336:351-352(2012)); XF134, XF136, XF140, XF141, XF15-1, XF15-1-1, TM1 (Jones, etal., Ann. Rev Phytopathol., 45:245-262 (2007)); and tonB1 (Summer etal., J. Bacteriol. 192:179-190 (2010)). Exemplary strains ofXanthomonas, which are susceptible to one or more of the disclosedbacteriophage isolates, and which may be useful for this inventioninclude EC12, Pres-4, and Jal-4 (provided by Dr. N. Wang, Univ. ofFlorida, Gainesville, Fla.), Noth 40, Ft. Basinger, and Block22, amongothers. Other Xanthomonad bacteria may also be utilized in view of theirsusceptibility to Xfas100 and/or Xfas300 bacteriophage.

As used herein, the term “isolation” is defined as separation andidentification of an organism from a solution containing a mixed cultureof organisms. Organisms able to be isolated can include viruses,bacteria, plant cells, or the like. Bacteriophage can be isolated asdescribed herein and known in the art. In one embodiment, generallaboratory methods for isolating bacteriophage may include but are notlimited to growth in cultured cells, bacteriophage assay, double agarmethod, and plaque assay, among others. The present invention provides amethod of isolating bacteriophage by a method involving overlaying atleast a first sample comprising different strains of bacterial hostcells together in order to isolate bacteriophage able to infect andpropagate within both host cell types.

The invention also provides a method of propagating a virus(bacteriophage) virulent to Xylella fastidiosa and/or Xa. Methods ofpropagating bacteriophages are known in the art, and can encompass anymethod capable of producing quantities of bacteriophage sufficient fortreating plant diseases. In one embodiment, propagating bacteriophagevirulent to X. fastidiosa and/or Xac can comprise growing bacteriophagein Xanthomonas bacteria, allowing the bacteriophage to propagate, andisolating bacteriophage particles from the culture.

Bacteriophage virulent to X. fastidiosa may be prepared using a softagar overlay method. High-titer phage plate lysates may be prepared, forinstance, by harvesting an overlay plate of X. fastidiosa strainTemecula or Xanthomonas strain EC-12 exhibiting confluent lysis,followed by maceration and clarification by centrifugation. After beingfilter sterilized, the resulting lysates can be stored at 4° C.Subsequently, high-titer phage lysates may be purified by isopycnic CsClcentrifugation, and extracted phage solution are dialyzed. CsCl-purifiedbacteriophage having a titer of, for instance, 1×10¹¹ PFU/ml can beobtained.

A preferred ratio of bacteriophage in plant tissue filtrates (PTFs) forfiltration is, for instance, 1 ml of PTF to 20 ml of the surrogate hostculture (actively growing culture of selected host), grown for 4 daysfor X. fastidiosa strain Temecula or for 4 hours for Xanthomonas strainEC-12.

The invention also provides a method of treating or reducing symptomsassociated with X. fastidiosa and/or Xa pathovars in a plant or plants.Typical Pierce's Disease (PD) symptoms include leaves becoming slightlyyellow or red along margins, respectively; eventually leaf margins maydry or die in its zones

One embodiment of the contemplated methods involves administering, to aplant infected with X. fastidiosa and/or Xa, bacteriophage(s) virulentto X. fastidiosa and/or Xa in a manner that will result in treatment ofthe plant. Treatment of plants for infection may be done by spraying,misting, dusting, injection, or any other method known in the art.Methods for formulating compositions for such applications are also wellknown in the art. For example, X. fastidiosa infects the vasculartissues of plants, and thus the invention as described herein maycomprise introducing via injection a purified population ofbacteriophage particles virulent to X. fastidiosa to a plant infectedwith X. fastidiosa such that the bacteriophage is able to infect andlyse the X. fastidiosa cells thereby treating the plant infection.However, one skilled in the art will recognize that other methods maysuccessfully be used, as well. Xa is a foliar pathogen and infects plantleaves, stems, and fruit naturally by rain splashing directly throughleaf stomata, or by way of wounds produced during strong winds or byinsects. Thus, in one embodiment, the present invention may compriseintroducing by spraying a composition comprising a purified populationof bacteriophage particles virulent to Xa to a plant infected with Xa.

As used herein, the terms “treatment,” “treating,” and “treat” aredefined as acting upon a disease, disorder, or condition with an agentto reduce or ameliorate the physiologic effects of the disease,disorder, or condition and/or its symptoms. “Treatment,” as used herein,covers any treatment of a disease in a host (e.g., a plant species,including those of agricultural interest, such as edible plants or thoseused to produce edible products, as well as ornamental plant species),and includes: (a) reducing the risk of occurrence of the disease in aplant, (b) impeding the development of the disease, and (c) relievingthe disease, i.e., causing regression of the disease and/or relievingone or more disease symptoms. “Treatment” is also meant to encompassdelivery of an inhibiting agent to provide an effect, even in theabsence of a disease or condition. For example, “treatment” encompassesdelivery of a disease or pathogen inhibiting agent that provides forenhanced or desirable effects in the plant (e.g., reduction of pathogenload, reduction of disease symptoms, etc.).

The invention also provides a plant disease biocontrol compositionformulated for delivery to a plant, the composition comprising at leastone carrier, and at least one bacteriophage that is virulent to Xylellafastidiosa and Xanthomonas species such as Xa.

The virulent bacteriophage to Xylella fastidiosa and/or Xanthomonasspecies such as Xa as an active ingredient in the composition of thepresent invention is also provided as one of bacteriophage selected fromthe group consisting of the Xfas100 phage type, such as Xfas101,Xfas102, Xfas103, Xfas104, Xfas105, Xfas106, Xfas107, Xfas108, Xfas109,and Xfas110, and/or the Xfas300 phage type, such as Xfas301, Xfas302,Xfas303, Xfas304, Xfas305, and Xfas306, wherein said phage type of theXfas103, Xfas106, Xfas302, Xfas303, Xfas304, and Xfas306, which havebeen deposited under ATCC Accession Numbers PTA-13096, PTA-13095,PTA-13098, PTA-13099, PTA-13100, and PTA-13097, respectively.

The virulent bacteriophage of the Xfas100 phage type as an activeingredient in the present invention displays at least one of thefollowing characteristics: (a) the bacteriophage has an activity of thecapable of lysing said Xylella fastidiosa and Xanthomonas bacteria, (b)the bacteriophage infects a cell by binding to a Type IV pilus, (c) thetailed bacteriophage exhibits long non-contractile tails with capsidranging from 55-77 mm in diameter, a morphology typical of Siphoviridaefamily, (d) the genomic size of bacteriophage is about 55500 bp to 56200bp and (e) the bacteriophage has an activity of preventing or reducingsymptoms associated with Pierce's Disease in a plant or plants.

The virulent bacteriophage of the Xfas300 phage type as an activeingredient in the present invention has at least one of thecharacteristics, wherein said characteristics is; (a) the bacteriophagehas an activity of the capable of lysing said Xylella fastidiosa andXanthomonas bacteria; (b) the bacteriophage infects a cell by binding toa Type IV pilus; (c) the group of a tailed bacteriophage exhibits shortnon-contractile tails with capsid ranging from 58-68 mm in diameter, amorphology typical of Podoviridae family; (d) the genomic size of thebacteriophage is about 43300 bp to 44600 bp; and (e) the bacteriophagehas an activity of preventing or reducing symptoms associated withPierce's Disease in a plant or plants. Virulent bacteriophage as anactive ingredient in compositions of the present invention furthercomprises at least one bacteriophage selected from the Xfas100 phagetype and/or the Xfas300 phage type, wherein said Xfas100 phage type isXfas103 and Xfas106 and/or said Xfas300 phage type is Xfas302, Xfas303,Xfas304, and Xfas306.

Bacteriophage virulent to Xylella fastidiosa and Xanthomonas species,such as Xa, used as an active ingredient in the composition of thepresent invention is also provided by a combination of phage, such as acocktail of two, three, four, five, six, or more virulent bacteriophageisolates or types, which may be provided simultaneously or sequentially,including with a carrier. The term “carrier” refers to a diluent,adjuvant, surfactant, excipient, or vehicle with which the phage isadministered. Such carriers can be sterile liquids, such as water andoils, including those of petroleum, animal, vegetable or syntheticorigin, such as peanut oil, soybean oil, mineral oil, sesame oil and thelike. Saline solutions, including phosphate solution such as sodiummonohydrogen phosphate, potassium dihydrogen phosphate and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable excipients may includestarch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,silica gel, sodium stearate, glycerol monostearate, talc, sodiumchloride, dried skim milk, glycerol, propylene glycol, water, ethanol,and the like.

A plant disease biocontrol composition, if desired, may also containminor amounts of wetting or emulsifying agents, or pH buffering agents.

Protective agents such as, but not limited to, casein basedformulations, flour-based formulations, sucrose, Congo red,N-propyl-gallete, and lignin-based formulations, can be added to a plantdisease biocontrol composition.

Phage concentration required for efficient disease control is notlimited, but for example can be from 1×10-1×10¹² PFU/ml, 1×10⁴-1×10¹¹PFU/ml or 1×10⁷-1×10¹⁰ PFU/ml.

Depending growing age of tree, the thickness of the stem, the size ofthe root, the dosage is adjusted appropriately. A plant diseasebiocontrol composition can be a dry product, a substantially dryproduct, a liquid product, or a substantially liquid product. In someembodiments, a dry or substantially dry product can be reconstituted ina liquid (e.g., water, etc.), and then applied to a plant. In otherembodiments, such a composition can be applied in dry or substantiallydry form, where liquid that is already present on the plant, isconcurrently applied to the plant, or that subsequently appears on theplant (e.g., by application, condensation, etc.) facilitates exposure ofthe bacteriophage to target bacteria. In another embodiment, such acomposition can be applied by spray, mist, or dust on the plant.

A plant disease biocontrol composition can take the form of a solution,a suspension, an emulsion, a powder, a tablet, and the like.

The timing of application of a plant disease biocontrol composition isnot limited, but may for instance be daily, weekly, or twice-weekly,monthly, bimonthly, or quarterly.

The present invention also provides an isolated bacteriophage that isvirulent to Xylella fastidiosa and Xanthomonas species, such as Xa andpathovars thereof.

The invention also provides an isolated bacteriophage as one ofbacteriophage selected from the group consisting of the Xfas100 phagetype, such as Xfas101-Xfas110, and/or the Xfas300 phage type, such asXfas301-Xfas306, and wherein Xfas103, Xfas106, Xfas302, Xfas303,Xfas304, and Xfas306, which have been deposited under ATCC AccessionNumbers PTA-13096, PTA-13095, PTA-13098, PTA-13099, PTA-13100, andPTA-13097, respectively.

Such a bacteriophage can be detected by confirming the capability offorming plaques on Xylella fastidiosa and/or Xanthomonas species.

DEPOSIT INFORMATION

A deposit of representative bacteriophage of each of strains Xfas103,Xfas106, Xfas302, Xfas303, Xfas304, and Xfas306, and a deposit ofrepresentative bacteria of X. anopodis EC-12, which are disclosed hereinabove and referenced in the claims, was made with the ATCC, located atP. O. Box 1549, Manassas, Va. 20108, USA. The date of deposit for theaccessions was Jul. 24, 2012 and the accession numbers for the depositedstrains are PTA-13096, PTA-13095, PTA-13098, PTA-13099, PTA13100,PTA13097, and PTA-13101, respectively. All restrictions upon the depositwill be removed upon the granting of a patent, and the deposit isintended to meet all of the requirements of 37 C.F.R. §1.801-1.809. Thedeposit will be maintained in the depository for a period of 30 years,or 5 years after the last request, or for the effective life of thepatent, whichever is longer, and will be replaced if necessary duringthat period.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments that are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Media, Culture Conditions and Bacterial Strains

This example describes the isolation, propagation, and the morphologicaland genomic characterization of bacteriophage virulent to X. fastidiosaand Xanthomonas species. The medium used in this study differs fromstandard medium used to grow X. fastidiosa, which allows rapid growthbut affects the ability of the bacteriophage to infect. PW broth mediumas modified by Sherald et al. (Plant Disease 67:849-852, 1983)designated PW-M, was used for growth of X. fastidiosa isolates, exceptthat the final bovine serum albumin content was 0.3% as modified by Hilland Purcell (Phytopathology 85(12):1368-1372, 1995). For solid medium(PW-MA) and soft agar, the PW-M broth was amended with 15 g/l and 7.5g/l, respectively, of Plant Cell Culture Tested agar (Sigma). Thecomplex medium TN broth (TNB) was used for routine maintenance of non-X.fastidiosa cultures. Solid medium (TNA) was identical, with theexception that it lacked KNO₃ and was supplemented with 20 g/L agar. Forsoft agar overlays, TN medium was amended with 7.5 g/L of agar (TNSA).For plating of plant extracts to obtain total bacterial counts, TNAmedium was amended with cycloheximide (40 μg/ml; TNAC). All cultureswere grown at 28° C. and liquid cultures were monitored at λ=600 nmusing Nephelo flasks. California X. fastidiosa isolates included in thestudy were Temecula (XF15), which is representative of X. fastidiosasubspecies fastidiosa, Ann1 (XF108), representative of X. fastidiosasubspecies sandyi, and Dixon (XF102), representative of X. fastidiosasubspecies multiplex (Hendson et al., Applied and EnvironmentalMicrobiology 67(2):895-903, 2001). Texas X. fastidiosa isolates includedone each from Platanus occidentalis (XF1), Helianthus annuus (XF5), Ivaannua (XF18), Ambrosia psilostachya (XF23), Ratibida columnifera (XF37),Vitis aestivalis (XF39), Vitis mustangensis (XF41), three isolates fromAmbrosia trifida var. texana (XF16, 40, and 43), two from Neriumoleander (XF93 and 95), and 15 from Vitis vinifera (XF48, 50, 52, 53,−54, 56, 58, 59, 60, 66, 67, 70, 71, 76, and 78). All isolates weresingle-colony purified by the streak isolation method, and stored at−80° C. after amending PW-M broth cultures to a final concentration of20% glycerol (v/v). X. fastidiosa isolates were confirmed at the speciesand subspecies level using polymerase chain reaction (PCR) analysis aspreviously described (Hernandez-Martinez et al., Plant Disease90(11):1382-1388, 2006). The MIDI Sherlock® Microbial IdentificationSystem that analyzes fatty acid methyl esters by gas chromatography(GC-FAME) was used to identify Xanthomonas species.

Example 2 Processing of Plant Samples and Isolation of Bacteria

Plant samples of Vitis vinifera, V. mustangensis, and weeds wereobtained from vineyards in Brazos County and Washington County, Texas.Rice (Oryza sativa) plant tissue and weeds from rice fields wereobtained from Jefferson County and Wharton County, Texas. Rice seedsamples were obtained from the Texas AgriLife Research Center inBeaumont, Tex. Samples from rose plants (Rosa spp.; Knock Out) andjalapeño (TAM-mild; Capsicum annuum) were obtained in Brazos County,Texas. To obtain plant extracts, 10 g of plant tissue were ground usinga mortar and pestle in 50 ml bacteriophage buffer (P-buffer; 50 mMTris-HCl pH 7.5, 100 mM NaCl, 8 mM MgSO₄), vortexed, and strainedthrough a double-layer of cheesecloth to remove large particles. Theextract was then dilution plated to both PW-M and TNAC for the isolationof X. fastidiosa and non-X. fastidiosa bacteria, respectively, andincubated at 28° C. Plates were evaluated for growth daily for up to 10days.

Example 3 Isolation, Purification and Titration of Bacteriophage fromPlant Samples

To obtain plant tissue filtrates (PTFs) the clarified plant extract wascentrifuged (10,000×g, 10 min at 4° C.), twice and the tissue extractwere filtered through a 0.22 μm filter (Supor, Pall Life Sciences). Thepresence of bacteriophage in PTFs was directly determined by spotting 10μl of a 10-fold dilution series on overlays of a panel of X. fastidiosaand Xanthomonas species hosts and observing for zone or plaque formationafter lawn development (6-7 days for X. fastidiosa isolates and 18 h forXanthomonas species isolates). Alternatively, bacteriophage wereenriched from the PTFs by adding 1 ml of each filtrate to 20 ml of anactively growing culture of an X. fastidiosa isolate (4 day culture;A₆₀₀=0.30) or an A₆₀₀=0.25 (4 h) culture of a Xanthomonas species host.After 72 h or 24 h of growth for the X. fastidiosa or Xanthomonasspecies enrichment, respectively, the cultures were centrifuged(10,000×g, 10 min at 4° C.) and filter sterilized (0.22-μm filter). Todetermine the presence of bacteriophage in the enriched supernatants, 10μl of a 10-fold dilution series was spotted on overlays of a panel of X.fastidiosa and Xanthomonas species hosts and observed for zone or plaqueformation. Five-day-old cultures of X. fastidiosa isolates grown onPW-MA were used to make host suspensions in PW-M broth (A₆₀₀=0.5),whereas 18-h cultures of Xanthomonas species isolates grown on mediumTNA were used to make suspensions in TN broth (A₆₀₀=0.5), and used tomake overlays. Soft agar overlays used to survey bacterial supernatantsfor bacteriophage activity were made by mixing 100 μl of the bacterialsuspension with 7 ml of molten PW-M or TN soft agar, pouring the mixtureon PW-MA or TNA plates, and allowing it to solidify and dry. Spottedoverlays showing either plaques or cleared zones formation were furtherinvestigated by plating as above, except that the PTF dilutions weredirectly mixed with individual host indicator suspensions beforeoverlaying. Individual plaques formed on either X. fastidiosa orXanthomonas species host overlays were excised, suspended in P-bufferand titered. This procedure was repeated three times to obtain a singleplaque isolate. High-titer lysates (1×10¹⁰ PFU/ml) were prepared byharvesting overlays of plates exhibiting confluent lysis with 5 ml ofP-buffer, macerating the soft agar overlay, clearing the lysate bycentrifugation (10,000×g, 15 min at 4° C.) and filter sterilizingthrough a 0.22-μm filter. Lysates were stored at 4° C.

Plant extracts were plated to both PW-M and to TNAC for selection of X.fastidiosa and to obtain total bacterial counts, respectively. Plantextracts from all plants assayed did not yield any evidence of X.fastidiosa isolates. However, the non-selective plating did yield alarge variety of bacterial colony types. Representative single coloniesof yellow pigmented bacteria were picked from plates and streak purifiedto obtain stocks. The stocks were used to make overlays to which thePTFs where spotted to observe for zone or plaque formation. Bacterialisolates Presidio-4, Jal-4, and EC-12 obtained from extracts of riceseed, jalapeño leaves, and rice tissue, respectively, were allidentified as Xanthomonas species using fatty acid methyl ester analysisby gas chromatography (GC-FAME) and were used as hosts for evaluation ofPTFs. Other Xa strains may be utilized as well, in view of the virulenceof one or more disclosed bacteriophage on such strains.

Dilutions of several PTFs produced plaques on isolates XF15, XF53, XF54,XF95, after 5 to 6 days of incubation, indicating that bacteriophageable to form plaques on these hosts were present in the plant tissue.Initial titers observed from extracts ranged from 5×10¹ to 7×10⁵PFU/gram of tissue. Since all PTFs showed the same pattern of productionon XF15, XF53, XF54, and XF95, strain XF15 was used as the host forplaque purification and production. The high titer found in thisnon-enriched PTF indicates that natural bacterial hosts associated withthe plant tissue can serve as host for bacteriophage, which can produceplaques on overlays of X. fastidiosa. Serial plating of the PTF yieldedindividual plaques that were uniform in size. Individual plaques wereexcised and plaque-purified three times using XF15 as the host to obtainclonal isolates. A culture dish of bacteriophage Xfas302 purified andincreased using Xylella fastidiosa host strain Temecula (1×10⁹ PFU(Plaque Forming Units/ml)) and titered on Xanthomonas species isolatePres-4 and EC-12 (both of 5×10⁷ CFU (Colony Forming Units)/ml),indicating that Bacteriophage Xfas302 propagated on X. fastidiosa strainXF15 (Jones et al., 2007) or X. fastidiosa Temecula strain (ATCC 700964)was able to form plaques on Xanthomonas species strain Pres-4 and EC12(EC12 deposited under ATCC Accession Number PTA-13101) providingevidence that bacteriophage propagated on X. fastidiosa can adsorb,replicate, and form plaques on Xanthomonas strains. Host range studiespresented below further substantiate these results.

Phages Xfas101-Xfas105 all produced small clear plaques on XF15 lawns,whereas phages Xfas301-Xfas305, produced large clear plaques on the samehost. TEM images of Xfas302-Xfas305 and Xfas101-Xfas104 are shown inFIG. 1 and FIG. 2, respectively. High titer lysates produced using XF15were used to obtain CsCl-purified preparations of each bacteriophage,which were used to conduct transmission electron microscopy (TEM)studies. Phages Xfas101-Xfas105 all exhibited long non-contractile tailswith capsids ranging from 55-64 nm in diameter, and thus were determinedto belong to the Siphoviridae family. Xfas301-Xfas305 all exhibitedshort non-contractile tails with capsids ranging from 58-65 nm indiameter, morphology typical of Podoviridae.

Example 4 Enrichment of Bacteriophage from Wastewater

Clarified wastewater samples were obtained from treatment facilities inthe Bryan, Tex. area. Samples were obtained from the Still Creek, CarterCreek, Turkey Creek, and Burton Creek facilities. Samples werecentrifuged (10,000×g, 10 min at 4° C.) twice and the tissue extractswere filtered through a 0.22-μm filter. Phages were enriched by adding 1ml of each filtrate to 20 ml of an actively growing culture of selectedhosts as described above. After 72 and 24 h of growth for the X.fastidiosa or Xanthomonas species enrichment, respectively, the cultureswere centrifuged and filter sterilized. The enriched filtrates werespotted to titer on overlays as described above.

Phages Xfas106-109 and Xfas306 were isolated from individually enrichedsamples obtained from the four wastewater treatment plants using hostEC-12 as the host. TEM studies of purified bacteriophage concentratesmorphologically identified bacteriophage Xfas306 as Podoviridae by ashort non-contractile tail with a capsid of 68 nm in diameter (FIG. 3),whereas phages Xfas106-109 isolated exhibited long non-contractile tailswith capsids of ˜77 nm in diameter (FIG. 3) characteristic ofSiphoviridae. Bacteriophage Xfas306 produced large clear plaques on bothhosts EC-12 and XF15, whereas phages Xfas106-109 produced small clearplaques on the same hosts (FIG. 3). Therefore, the method used in thisexperiment enabled isolation of X. fastidiosa bacteriophage fromenvironmental samples.

Example 5 CsCl-Purification

Filter-sterilized bacteriophage suspensions were concentrated bycentrifugation (90,000×g for 2.5 h at 5° C.) using a Type 60Ti rotor ina Beckman L8-70M ultracentrifuge. Pellets were resuspended in P-bufferand treated with DNase I and RNase A (Sigma) at a final concentration of1 μg/ml at 25° C. for 2 h. CsCl was added to the bacteriophagesuspension at a final concentration of 0.75 g/ml and centrifuged(300,000×g for 18 h at 5° C.) in a VTi 65.2 rotor. The visiblebacteriophage band was extracted using an 18-gauge syringe needle anddialyzed against P-buffer amended to 1 M NaCl overnight at 4° C. andtwice for 4 h at 25° C. against P-buffer to obtain a suspension of1×10¹¹ PFU/ml. The CsCl-purified bacteriophage was stored at 4° C.

Example 6 Transmission Electron Microscopy

Electron microscopy of CsCl-purified bacteriophage (1×10¹¹ PFU/ml) wasperformed by diluting with P-buffer and applying 5 μl onto a freshlyglow-discharged formvar-carbon coated grid for 1 min. Grids were thenwashed briefly on de-ionized water drops and stained with 2% (w/v)aqueous uranyl acetate. Specimens were observed on a JEOL 1200EXtransmission electron microscope operating at an acceleration voltage of100 KV.

Example 7 Efficiency of Plating and Host Range

The efficiency of plating (EOP) was obtained by calculating the ratio ofthe bacteriophage plaque titer obtained with the heterologous(non-propagating) host to that obtained on the homologous (propagating)host. Bacteriophage stocks were titered on either X. fastidiosa orXanthomonas species host using the appropriate medium by mixing 100 μlbacteriophage stock dilutions with individual host indicator suspensions(A₆₀₀=0.5) in soft agar (7 ml) before overlaying on solid medium.

Studies comparing the EOP of Xfas phages are shown in Table 1. The EOPfor phages isolated from plant samples propagated using Xanthomonasspecies strain EC-12 and then titered using X. fastidiosa strain XF15 asthe host, ranged from 1×10⁻¹ to 1×10⁻³, with similar results seen whenbacteriophage propagated using strain XF15 were then titered using EC-12as the host. Similar studies with phages isolated from wastewaterfiltrates and propagated on strain EC-12 exhibited EOPs ranging from1×10⁻¹ to 5×10⁻¹. EOPs of 1×10⁻¹ to 3×10⁻¹ were obtained when phagesXfas106-109 were propagated on strain XF15 and plated on host EC-12,indicating that, while DNA restriction and modification barriers mayexist, phages propagated in fast-growing strain EC-12, in one day, canadsorb, replicate and form plaques on X. fastidiosa, a process which cantake up to 10 days on X. fastidiosa alone.

TABLE 1 Efficiency of Plating for Xfas Phages Propagated on X.fastidiosa or Xanthomonas species hosts Bacteriophage Propagated →Plated Propagated → Plated Designation XF15 → EC-12 EC-12 → XF15 Xfas1013.00E−02 5.00E−02 Xfas102 3.33E−02 5.00E−02 Xfas103 1.00E−02 3.33E−03Xfas104 4.00E−01 5.00E−02 Xfas105 1.00E−01 1.00E−01 Xfas106 1.00E−012.50E−01 Xfas301 5.00E−03 1.00E−02 Xfas302 1.00E−01 5.00E−04 Xfas3032.67E−03 1.00E−03 Xfas304 1.00E−02 5.00E−03 Xfas305 1.00E−02 5.00E−02Xfas306 3.33E−01 3.00E−01

Lawns of the host were made by overlaying plates of the appropriatemedium, PW-M (for XF15) or TNA (for EC-12) with the homologous soft agarseed with individual host. High titer lysates (1×10⁹ PFU/ml) ofindividual bacteriophage preparations were then spot titered on to theindividual lawns by spotting 10 μl of a 10-fold dilution series onoverlays of the X. fastidiosa or Xanthomonas species hosts. Afterincubation of plates at 28° C. for the appropriate times, (24 h forEC-12 or 5-7 days for XF15) plates were evaluated for zones and plaqueformation.

Initial host range studies shown in Table 2 indicate that all phageswhich were able to form plaques on X. fastidiosa host XF15 also formedplaques on host EC-12, whereas hosts Jal-4 and Pres 4 exhibitedinsensitivity to most of the siphophages. Reasons for resistance rangefrom lack of adsorption or other post adsorption mechanism such asbacteriophage-genome uptake blocks, superinfection immunity, restrictionmodification, and clustered regularly interspaced short palindromicrepeats (CRISPRs).

TABLE 2 Host Range of Xfas phages* Hosts Temecula Ann1 Dixon Phages(XF15) EC-12 Jal 4-1 Pres 4 (XF108) (XF102) Xfas101 S S S R S S Xfas102S S S R S S Xfas103 S S R R S S Xfas104 S S R R S S Xfas105 S S S R S SXfas106 S S R R S S Xfas107 S S R R S S Xfas108 S S R R S S Xfas109 S SR R S S Xfas301 S S R R S S Xfas302 S S S S S S Xfas303 S S S S S SXfas304 S S S S S S Xfas305 S S S S S S Xfas306 S S R R S S *EC-12 hostfor propagation of phage used in testing. S = ability to form clearplaques on indicated host; R = not able to form plaques on indicatedhost.

Example 8 Preliminary Identification of Xfas Adsorption Sites

Based on the observation that X. fastidiosa phages obtained or enrichedfrom either plant tissue or wastewater samples formed plaques on X.fastidiosa it was of interest to determine if cell surface componentscould serve as adsorption sites. Known adsorption sites for phagesinclude surface proteins such as OmpA and OmpF, the core and O-chain ofthe bacterial LPS in Gram-negative bacteria, sex and type IV pili, andflagella. The wild type and a derivative mutant with a deletion of thepilA, resulting in a derivative devoid of type IV pili, were evaluatedas hosts for Xfas phages. All bacteriophages tested formed plaques onthe XF15 wild type strain but not the XF15ΔpilA mutant. Resultssuggested that type IV pili may be a primary site of attachment for Xfasphages.

Based on results obtained with XF15-ΔpilA it was of interest todetermine if pilA deletion mutants of Xanthomonas species strain EC-12would become insensitive to Xfas phages Xfas103, Xfas106 Xfas302,Xfas303 Xfas304 and Xfas306. Strain EC-12Δ pilA was insensitive to thephages in plate titer assays, and in an adsorption experiment with phageXfas303 no adsorption to the host was observed. TheEC-12ΔpilA-complemented in trans for the pilA was sensitive to alltested phages. This further demonstrated that type IV pili are a primarysite of attachment for phages as observed for X. fastidiosa.

Example 9 Bacteriophage DNA Isolation and Genome Sequencing

Bacteriophage genomic DNA was prepared from 10-20 ml offilter-sterilized, high-titer (>1×10⁹ PFU/ml) CsCl-purifiedbacteriophage lysates using a modified form of the Promega Wizard DNAclean-up kit (Promega). Briefly, 10-20 ml of bacteriophage lysate wasdigested with 10 μg/ml each of DNase I and RNase A (Sigma) at 37° C. for30 min and precipitated in the presence of 10% (w/v) polyethylene glycol8000 and 1 M NaCl for 16-20 h at 4° C. The precipitate was centrifugedat 10,000×g, 4° C. for 10 min and the pellet resuspended in 0.5 ml ofP-buffer. One ml of the DNA purification resin supplied with the Wizardkit was added to the resuspended bacteriophage, loaded onto a minicolumnand washed with 2 ml of 80% (v/v) isopropanol. DNA was eluted from theresin by addition of 100 μl of water pre-heated to 80° C. followedimmediately by centrifugation of the minicolumn. DNA integrity wasverified by running on a 0.8% agarose gel and staining with ethidiumbromide and DNA was quantified by band densitometry. Bacteriophagegenome size was estimated by pulsed-field gel electrophoresis (PFGE)analysis of genomic DNA on a 1% agarose gel (Pulsed-Field agarose,BioRad) and comparison to a size marker (MidRange Marker I, NEB).

Phages were sequenced using “454” pyrosequencing (Roche/454 LifeSciences, Branford, Conn., USA, at Emory GRA Genomics Core: Emory Univ.,Atlanta, Ga.). Bacteriophage genomic DNA was prepared from bacteriophageisolates as described above and mixed in equimolar amounts to a finalconcentration of ca. 100 ng/μl. The pooled DNA was sheared, ligated witha multiplex identifier (MID) tag specific for each of the four pools andsequenced by pyrosequencing using a full-plate reaction on a Roche FLXTitanium sequencer according to the manufacturer's protocols. The pooledbacteriophage DNA was present in two sequencing reactions. The reactioncontained genomic DNA representing 39 genomic elements totaling ca.3,331 kb of genomic sequence, and the sequencing run yielded 987,815reads with an average length of 405 bp, providing a total of 120-foldcoverage for the entire pool. The trimmed FLX Titanium flowgram outputscorresponding to each of the four pools were assembled individuallyusing the Newbler assembler version 2.5.3 (454 Life Sciences) byadjusting settings to include only reads containing a single MIDidentifier per assembly. The identity of individual contigs wasdetermined by PCR using primers generated against contig sequences andindividual bacteriophage genomic DNA preparations as template; thegeneration of the expected size product from a bacteriophage DNAtemplate was used to match individual phages to their contigs.Sequencher (Gene Codes Corporation) was used for sequence assembly andediting. Protein coding regions were predicted using Genemark(opal.biology.gatech.edu/GeneMark/gmhmm2_prok.cgi) and manually editedin Artemis (www.sanger.ac.uk/Software/Artemis/) (Lukashin et al.,Nucleic Acids Research 26(4):1107-1115, 1998; Rutherford et al.,Bioinformatics 16(10):944-945, 2000). DotPlots were generated usingDOTTER (Brodie et al., Bioinformatics 20(2): 279-281, 2004). Predictedproteins were compared to proteins in the GenBank database using BLAST(www.ncbi.nlm.nih.gov/blast/Blast.cgi). Conserved domains, lipoproteinprocessing signals and transmembrane domains (TMDs) were identified withInterProScan (www.ebi.ac.uk/Tools/webservices/services/interproscan),LipoP (www.cbs.dtu.dk/services/LipoP/), and TMHMM(www.cbs.dtu.dk/services/TMHMM/), respectively.

TABLE 3 TABLE 3. Genomic size of Xfas Phages. Family ATCC Genomic of SEQAccession Size Identity Morphology Phage ID Numbers (bp) (bp) Dice score(% identity over entire genome of Xfas103) Xfas100 Siphoviridae Xfas10111 56,132 56,144 100.01 Types Xfas102 12 56,132 56,144 100.01 Xfas103 13PTA-13096 56,147 56,147 100.00 Xfas104 14 56,144 56,144 100.00 Xfas10515 56,144 56,144 100.00 Xfas106 16 PTA-13095 55,601 31,026 55.53 Xfas10717 Xfas110 18 56,134 56,144 100.01 Dice score (% identity over entiregenome of Xfas303) Xfas300 Podoviridae Xfas301 19 44,443 33,254 75.25Types Xfas302 20 PTA-13098 44,521 33,347 75.39 Xfas303 21 PTA1309943,940 43,940 100.00 Xfas304 22 PTA-13100 43,869 1,933 4.40 Xfas305 2343,324 43,940 100.71 Xfas306 24 PTA-13097 43,745 32,886 75.01 Dice Score= ((2 × identity)/(Sequence length of both phages)) × 100

Example 10 Genomic Analysis of Xfas Phages and Description of theXfas100 and Xfas300 Phage Types

The phages isolated for their ability to attack Xanthomonas EC-12 and X.fastidiosa and subspecies that all require the type IV pili forinfection and are all virulent, in that no lysogenic colonies can beisolated from infections and no genes associated with temperate lifestyle (repressor, integrase) are found in the genome sequences. Thephages can be further classified in two phage types, as defined byCasjens et al. (Research in Microbiology, 159:340-348, 2008).

(1) Xfas100 phage type: The Xfas100 phage type is comprised of virulentSiphophages (ICTV Siphoviridae) of Xanthomonas and Xylella, theprototypes of which are the phages Xfas101, Xfas102, Xfas103, Xfas104,Xfas105, Xfas106, Xfas107, Xfas108, Xfas109, and Xfas110 (Table 12) andfurther examples of which are listed in Table 3 as any phage designated“Xfas1nn,” where n is any number (referred to as the Xfas100 series).This flexible nomenclature system is necessary because it is anticipatedthat further variants of the Xfas1nn phage type will be isolated.Xfas100-type phages are siphophages, are virulent in life-style, andrequire the type IV pili for infection of Xylella and Xanthomonasspecies. Xfas100-type phages have icosahedral capsid heads measuringapproximately 55-77 nm in diameter and flexible tails of approximately200-262 nm in length; both dimensional values are as determined withinthe standard precision of negative-stain electron microscopy (see FIGS.2 and 3). The Xfas100 series viral DNA has cohesive (cos) endscharacterized by single-stranded DNA overhangs (Casjens, et al., MethodsMol Biol 502:91-111, 2009), which is important for phages to be used inantibacterial applications because cos DNA packaging avoids thegeneration of generalized transducing particles that would potentiatethe transfer of pathogenesis determinants. The Xfas100 genome has acharacteristic overall organization (see FIG. 4) with the genes arrayedin two divergent gene clusters, A_(L) and A_(R) and B_(L) and B_(R). TheXfas100 phage type is further distinguished by the fact that theessential structural and lysis genes of the phage are grouped inrightward gene cluster B_(L). The Xfas100 series phage type is alsodistinguished by encoding its own single-molecule DNA polymerase(Xfas103gp71 and Xfas106gp66), primase (Xfas103gp76 and Xfas106gp71) andhelicase (Xfas103gp69 and Xfas106gp64).

(2) Xfas300 phage type: The Xfas300 phage type is comprised of virulentpodophages (ICTV Podoviridae) of Xanthomonas and Xylella, the prototypesof which are the phages Xfas301, Xfas302, Xfas303, Xfas304, Xfas305, andXfas306, and further examples of which are listed in Table 3, and refersto any phage with the designation “Xfas3nn” where n is any number(referred as Xfas300 series). This flexible nomenclature system isnecessary because it is anticipated that further variants of the Xfas300phage type will be isolated. Xfas300-type phages have icosahedral capsidheads measuring approximately 58-68 nm in diameter; this dimensionalvalue is as determined within the standard precision of negative-stainelectron microscopy (see FIGS. 1 and 3). The Xfas302-306 genome encodesa single-subunit RNA polymerase located adjacent to the structuralprotein region. The Xfas300 series genome has a characteristic overallorganization (see FIG. 5) with the genes arrayed on one strand,including the replication, structural and lysis genes of the phage. TheXfas300 phage type is also distinguished by encoding its ownsingle-molecule DNA polymerase (Xfas302gp18, Xfas303gp17, Xfas304gp17and Xfas306gp17), single-subunit RNA polymerase (Xfas302gp31,Xfas303gp28, Xfas304gp27 and Xfas306gp30, respectively and helicase(Xfas302gp15, Xfas303gp14, Xfas304gp15 and Xfas306gp14 (see FIG. 5 forschematic of phage genome).

Example 11 Movement, Challenged and Protection Studies in GrapevinesUsing Bacteriophage Xfas304

Bacteriophage Xfas304, is a member of the family Podoviridae, isolatedfrom environmental samples that has a host range that includes both X.fastidiosa and Xanthomonas species. In the studies presented here, themovement and persistence of Xfas304 was determined in grapevines in theabsence of a sensitive host, in order to determine whether treatment ofa plant with bacteriophage may prevent subsequent infection by X.fastidiosa. Additionally, grapevines that were first inoculated with X.fastidiosa were then challenged 4 weeks post-pathogen-inoculation withbacteriophage Xfas304, to determine if the bacteriophage could controlthe development of Pierce's Disease therapeutically.

For the preventative studies, grapevines were inoculated with 40 μl of abacteriophage Xfas304 suspension (1×10¹⁰ PFU/ml) and then challenged 4weeks post-bacteriophage-inoculation with X. fastidiosa. Bacterial X.fastidiosa suspensions used for inoculation were adjustedspectrophotometrically (A₆₀₀=0.4; 1×10⁹ CFU/ml). Individual cordons wereinoculated between the second and third node on opposite sites (twopoints/cordon) with 40 μl of the bacterial suspension using the needleinoculation technique as described by Hopkins (Plant Dis. 89:1348-1352,2005). Control vines were mock inoculated with phosphate buffer at thesame point of inoculation of the above.

The results indicated that bacteriophage Xfas304 can be used to treatand prevent Pierce's Disease caused by X. fastidiosa subspeciesfastidiosa in grapevines. Thus, bacteriophage Xfas304 and other virulentXylella-Xanthomonas phages identified from these studies have potentialuse in the protection and treatment of plants against diseases caused byother X. fastidiosa subspecies and Xanthomonas species.

Bacteria used in the study included X. fastidiosa strains Temecula(XF15) and XF54, associated with Pierce's Disease of grapevines inCalifornia and in Texas, respectively. Cultures of X. fastidiosa weremaintained on PW-M agar medium (Summer et al., J Bacteriol 192(1):179-190, 2010) at 28° C. for 5-7 days. Five-day-old cultures of the X.fastidiosa isolates grown on PW-MA were used to make bacterialsuspensions in phosphate buffer (0.125 M, pH 7.1) for vine inoculations.

Dormant V. vinifera cv. Cabernet Sauvignon clone 08 on 1103P rootstockwere purchased from Vintage Nurseries (Wasco, Calif., USA), Vines wereplanted in 7-gallon pots using 101 Sunshine Mix 1 (Sun Gro Horticulture,Vancouver, British Columbia, Canada). Plants were grown in a greenhouseon a 16-h light (26° C., 300-400 μE/m²·s)/8-h dark (18° C.) cyclesupplemented with illumination from sodium vapor lamps. Plants wereirrigated every other day with tap water. Every 15 days, the vines werefertilized with Peter's General Purpose 20-20-20 fertilizer andmicronutrients. Plants were progressively pruned to provide uniformplants as follows: upon producing two unbranched solitary shoots of100-150 cm, two shoots were pruned to 80 cm. Lateral shoots and budswere removed. Two cordons were staked and allowed to grow until eachcordon was ˜2.5-2.75 m in length before vines were used for theabove-experiments.

Standard qRT-PCR line plots were obtained for X. fastidiosa strains XF15and XF54, as well as for bacteriophage Xfas304, all of which had R²values of greater than 0.9 and efficiencies of 157%, 130%, and 123%,respectively. Quantitative assessment of duplicate cordons fromtriplicate samples of XF15 and XF54 showed distribution of the pathogensthroughout all segments assayed, with typical Pierce's Disease (PD)symptoms visible, such as leaves become slightly yellow or red alongmargins, respectively, and eventually leaf margins dry or die in itszones by week 8 post-inoculation (FIG. 6). In vines inoculated withbacteriophage Xfas304, in the absence of a permissive host, aprogression in the distribution of bacteriophage at weeks at 2, 4, and 6weeks post-inoculation was observed, with a decline between weeks 8-12and no vine symptoms.

Example 12 Grapevine Inoculation with Bacteria and Bacteriophage

For therapeutic evaluation of bacteriophage treatment, 15 vines (twocordons each) were inoculated with X. fastidiosa strains XF15 or XF54.Bacterial suspensions used for inoculation were adjustedspectrophotometrically (A₆₀₀=0.4; 1×10⁹ CFU/ml). Average of qRT-PCRresults from three segments (e.g. 1/1a, 1/1b, 1/1c) with similarlocations from triplicate vines was used to determine the CFU/gram planttissue (gpt) and PFU/gpt. Individual cordons were inoculated between thesecond and third node on opposite sites (two points/cordon) with 40 μlof the bacterial suspension using the needle inoculation technique asdescribed by Hopkins (Plant Dis. 89:1348-1352, 2005). Control vines weremock inoculated with phosphate buffer following the same protocol. Fourweek post-inoculation with the pathogen, the 15 vines from eachtreatment were challenged with 40 μl of a bacteriophage Xfas304suspension (1×10¹⁰ PFU/ml) using the same inoculation protocol andtechnique. Vines were scored for symptom development twice weekly for 12weeks and assayed in triplicate for X. fastidiosa and bacteriophage atthe time of inoculation, and at 8, 10, and 12 weeks, as described below.To determine if bacteriophage could act in a preventative manner, ninevines (two cordons each) were inoculated with 40 μl bacteriophageXfas304 using the same inoculation protocol and inoculation technique asthe above. At four weeks post-bacteriophage inoculation, the vines werechallenged with 40 μl (A₆₀₀=0.4; 1×10⁹ CFU/ml) of strain XF15 using thesame inoculation protocols as the above.

To evaluate the bacterial and bacteriophage movement in the grapevine,15 vines each were inoculated with either XF15 or XF54 and 24 vines wereinoculated with only bacteriophage Xfas304 using the same inoculationprotocols as the above. Vines inoculated with XF15 or XF54 were assayedin triplicate immediately after inoculation and at weeks 8, 10, and 12post-inoculation. Vines inoculated with bacteriophage were assayed intriplicate immediately after inoculation and every two weeks for 12weeks. Methods for assay are described below.

To determine how bacteriophage would affect pathogen populations anddisease development in vines, X. fastidiosa inoculated vines werechallenged with bacteriophage Xfas304 at four weeks post-pathogeninoculation. At 8 weeks post inoculation with XF15, vines challengedwith Xfas304 at week 4 showed no PD symptoms and the bacterialpopulations were one to three logs lower in bacteriophage challengedvines as compared to non-challenged vines. The non-bacteriophagechallenged plants showed PD symptoms (FIG. 7, column 2), whereas thebacteriophage challenged vines showed no PD symptoms after week 5 (FIG.7, column 6). During weeks 8 through 12 post-XF15 inoculation (weeks 4through 8 post-Xfas304 challenge), no PD symptoms were observed inbacteriophage-challenged vines and XF15 populations continued to declineto almost non-detectable levels as compared to non-bacteriophagechallenged vines.

A quantitative evaluation of the bacteriophage population in thepresence and absence of an introduced host (XF15) indicated that thebacteriophage were able to replicate in sensitive hosts growing in thevines and declined in the absence of a sensitive hosts. Experiments withstrain XF54 challenged with Xfas304 at 4 weeks post-pathogen inoculationshowed similar results to that observed for XF15 challenged vines. TheXF54 population in vine extracts, as measured by CFU/ml of extract,declined from weeks 8 through 12 in bacteriophage challenged vines ascompared to that observed in non-bacteriophage-challenged vines. Atweeks 8 through 12 post XF54 inoculation (weeks 4 through 8,post-Xfas304 challenge), no PD symptoms were observed inbacteriophage-challenged vines (FIG. 7, column 7). The bacteriophagepopulation increased over the post challenge period in the presences ofXF54 and decreased in the absence of a host, again indicating that thebacteriophage was able to replicate in sensitive hosts when present invines. A summary of the challenge study is presented in FIG. 7, showingthat in XF15 or XF54 inoculated vines challenged with bacteriophageXfas304 (week 4 post-pathogen inoculation) no additional PD symptomswere observed after week 5 (FIG. 7, columns 6 and 7), whereas symptomdeveloped through week 9 and 10 in non-bacteriophage challenged vinesinoculated with strain XF15 or XF54, respectively (FIG. 7, columns 2 and3).

Additional studies were conducted with vines inoculated withbacteriophage Xfas304 and then challenged with XF15 at week 4post-bacteriophage inoculation to determine the protective(prophylactic) potential of the bacteriophage treatment. At weeks 4 and8 post-challenge with XF15 vines showed no PD symptoms (FIG. 7, column8), whereas non-phage treated vines developed symptoms (FIG. 7, column2). The bacteriophage population increased from week 8 to 12 postchallenge period in the presences of XF15 and decreased in the absenceof a host.

These results confirm that bacteriophage treatment prevents or reducesPD symptoms by X. fastidiosa in a plant and that phage treatment causesno adverse effects to a plant.

Example 13 Sample Collection and Processing

Duplicate cordons from each vine were divided into 5-6 segments andsegments were numbered from bottom to top. Each segment was homogenizedusing a PRO250 homogenizer with 20×115 mm generator (PRO Scientific, CT,USA) in 15 ml of P-buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 8 mMMgSO₄), filtered through sterile cheesecloth (Fisher Scientific, USA) toremove plant tissue debris. For assaying bacteriophage, the filtrate wascentrifuged (10,000×g for 15 min) and filter sterilized. The filtratewas used for bacteriophage DNA extraction. The same protocol was usedfor bacterial assays, except the pellet was resuspended into 1 ml ofMilli-Q water for bacterial DNA extraction.

Example 14 Propidium Monoazide (PMA) Treatment of Samples

The PMA protocol as described by Nocker (J Microbiol Meth 67:310-20,2006) was used to exclude dead cells of X. fastidiosa from assays usedto develop standard curves for assays and for assaying of vine tissueextracts. Briefly, PMA (Biotium Inc., Hayward, Calif.) was dissolved in20% dimethyl sulfoxide (Sigma-Aldrich, Germany) to yield a stocksolution of 20 mM and stored in the dark at −20° C. A volume of 1.25 μlof the PMA stock solution was added to 500 μl X. fastidiosa cellsuspensions (A₆₀₀=0.4; 1×10⁹ CFU/ml) or extracts from control andinoculated vines. Preparations were incubated in clear microcentrifugetubes in the dark for 5 min with repeated inversion. Followingincubation, the microcentrifuge tubes were placed on ice and exposed toa 650-W halogen light source (Ushio, USA) at a distance of 20 cm for 1min. The tubes were swirled briefly by hand every 15 s and invertedafter 30 s of illumination to ensure complete cross-linking of theavailable DNA and the conversion of free PMA to hydroxylamino propidium.After photo-induced cross-linking, viable cells were collected bycentrifugation at (12000×g for 2 min at 25° C.) and washed with 500 μlsterile distilled water and resuspended Mili-Q water for DNA extraction.

Bacterial DNA was extracted from PMA treated cell preparations and vineextracts using a ZR Fungal/Bacterial DNA Miniprep (Zymo Research, USA)as per the manufacturer's instructions.

Bacteriophage DNA from control preparations and from plant extracts wasextracted using Wizard DNA Clean-up system (Promega, Wis., USA) withmodifications as described by Summer (Methods Mol. Biol. 502:27-46,2009).

Example 15 Detection of X. fastidiosa and Bacteriophage Xfas304 UsingReal Time-PCR

A SYBR-green based Real Time-PCR (RTPCR) was performed on the 7500Real-Time PCR System (Applied Biosystems, CA, USA) using the X.fastidiosa-specific primers INF2 5′-GTTTGATTGATGAACGTGGTGAG-3′ (SEQ IDNO:1) and INR1 5′-CATTGTTTCTTGGTAGGCATCAG-3′ (SEQ ID NO:2) designed forthe gyr B (Bextine and Child, FEMS Microbiology Letters 276: 48-54,2007), and bacteriophage Xfas304-specific primers 304-PrimF5′-AAGAAGCGTGGTTTGTTTGC-3′ (SEQ ID NO:3) and 304-PrimR5′-CTACCGGCTTCCCTAACTCC-3′ (SEQ ID NO:4) designed for the DNA primasegene. A master mix was made using 10 μl of Express SYBR GreenER SuperMix(Invitrogen), 0.4 μl of both primers (at a concentration of 10 μM), 8.56μl of sterile molecular grade water, 0.04 μl of ROX reference dye(Invitrogen), and 1 μl DNA template per reaction. Standardizedconditions were used for all reactions with an initial denaturing stepof 3 min at 95° C., followed by 40 cycles of the following parameters:95° C. for 30 s, 55° C. for 30 s, and 72° C. for 30 s. At the end of thePCR, the temperature was increased from 72 to 99° C. at a rate of 0.5°C./10 s, and the fluorescence was measured every 10 s. Each DNA samplewas analyzed in triplicate. As a positive control, DNA was extractedfrom X. fastidiosa cells and from bacteriophage Xfas304 lysates usingmethods described the above. Cycle threshold (Ct) values, describing thePCR cycle number at which fluorescence rises above the base line, weredetermined using the software package provided by the AppliedBiosystems.

To determine the Standard curve for absolute quantification, 1-mlvolumes of XF15 and XF54 cell suspensions of 1×10⁸ CFU/ml were treatedusing the PMA protocol. Bacterial DNA was extracted as described above,serially diluted from 1×10⁻¹ to 1×10⁻⁵ and subjected to the real-timePCR assay described above. Similarly, bacteriophage DNA was extractedfrom 1 ml volume of 1×10⁹ PFU/ml bacteriophage Xfas304 as describedabove, diluted from 1×10⁻¹ to 1×10⁻⁶ and subjected to the real-time PCRassay. Three replicates of each sample for X. fastidiosa andbacteriophage Xfas304 were used to produce the standard curves. Standardcurves were constructed by plotting Ct values generated from real-timePCR against X. fastidiosa DNA concentrations (Log DNA conc./μl asdetermined by A₂₆₀). The efficiency (E) was calculated as follows:E=10^((−1/slope))−1 (Klein et al., Electrophoresis 20:291-299, 1999).

Example 16 Lysogen Formation Assay Studies

To assay for phage lysogen-formation, survivors of phage infection weretested for the presence of prophages. For each phage, bacteria wereinfected with at an input MOI of ˜3 and plated in a soft agar overlay.Plates were monitored for colony growth (10-15 days for X. fastidiosastrain Temecula and 2-3 days for Xanthomonas strain EC-12). Individualcolonies that emerged were picked, purified (three times) and re-testedfor phage sensitivity by spotting dilutions of the same phage in a softagar overlay. Primer pairs specific to the Xfas103 and Xfas106 helicasegene, or Xfas303 and Xfas304 primase gene (Table 5) were then used totest for the presence of prophage sequences in the phage-insensitiveisolates. Wild type bacterial DNA was used as negative control and wildtype bacterial DNA spiked with phage DNA served as positive controls.

To test whether evidence could be found for abortive lysogeny (i.e., theestablishment of repression), we followed the procedure of Gill et. al(Gill J. J., et al., J. Bacteriol., 193:5300-5313 (2011)), exceptreversibly bound phage were removed by three successive washes. Liquidcultures of logarithmically growing Xanthomonas strain EC-12 werecultured to an OD600 of ˜0.3-0.5. One ml aliquots were pelleted bycentrifugation and resuspended in 0.20 ml of phage lysate (harvested inTNB) or sterile TNB. After a 30 min incubation at 25° C. cell-phagemixtures were centrifuged and the supernatant removed and titered todetermine adsorbed phage. In preliminary experiments it was determinedthat phage were reversibly bound, which affected the MIOactualcalculation (Kasman, L. M., et al., J. Virol., 76:5557-5564 (2002)). Tocircumvent this problem and to obtain an accurate MOIactual, cells wereresuspended in sterile TNB, allowed to incubate for 5 min at 25° C.,centrifuged and supernatants removed. This procedure was repeated threetimes to remove unbound phage. Each supernatant was titered to determinePFU. Cell pellets were resuspended in 0.20 ml of sterile TNB, seriallydiluted and plated to enumerate the bacterial survivors remainingfollowing phage exposure. From these data, the MOIactual, i.e., theratio of the number of adsorbed phage to the number of CFU in thephage-free controls) was calculated. These MOIactual values were used tocalculate the predicted proportion of uninfected cells using the Poissondistribution. This experiment was replicated three times, using bothXfas103 and Xfas303.

Lysogeny.

To examine the potential for lysogeny, 40 phage-insensitive isolates ofX. fastidiosa strain Temecula and Xanthomonas strain EC-12 each wererecovered following a challenge by phage Xfas103, Xfas106, Xfas303 orXfas304. PCR using phage specific primers did not detect the presence ofphage lysogens in resistant isolates, indicating resistance was not dueto lysogeny. Additionally, the potential for abortive lysogeny wasexamined using infection at a high MOI and measuring survival (Gill et.al (2011)). As shown in Table 4, following infection, there was nosignificant difference between predicted and actual survivors,indicating phage infection at a high MOI did not lead to theestablishment of repression. Together, these results indicate there isno evidence for lysogeny or repression, supporting the conclusion thatthe four phages are virulent.

TABLE 4 Predicted bacterial survivors based on MOIactual compared tomeasured bacterial survivors of Xanthomonas strain EC-12 followingexposure to phage Xfas 103 or Xfas 303a. Predicted Measured Fold excess% % of Replicate surviving surviving survivors vs. No. MOI_(actual)cells cells prediction Xfas 103 1 6.51 0.15 0.12 0.8 2 5.57 0.38 0.250.65 3 5.99 0.30 0.24 0.80 Xfas 303 1 5.40 0.45 0.38 0.80 2 5.39 0.450.49 1.08 3 5.52 0.40 0.37 0.92 a Predicted survivors were calculatedfrom the Poisson distribution for the measured MOIactual. Data shown arefrom three independent replicate experiments.

Example 17 Phage Cocktail Protection and Prevention Studies

Bacterial Strains, Phages, and Inoculum Preparation:

Bacterial isolates used in the study were X. fastidiosa strains Temecula(XF15) and XF54 (see Example 3). Cultures of X. fastidiosa weremaintained on PW-M as described in Example 1. XF15 and XF54 inocula wereprepared as described in Example 11. High-titer phage lysates ofXfas303, Xfas304, Xfas103 and Xfas106 (1×10¹⁰ PFU/ml) were prepared andtitered as described in Example 3. The phage cocktail was prepared bymixing each of the four phages to obtain a final concentration of 1×10¹⁰PFU/ml for each phage in the cocktail.

Grapevine Inoculation with Bacteria and Phage Cocktail:

Grapevines were inoculated with either X. fastidiosa strain XF15 or XF54to evaluate bacterial movement in the grapevine. Grapevines were assayedin triplicate immediately after inoculation (0 min) and at 8 and 12weeks post-inoculation. Additionally, grapevines inoculated with XF15 orXF54 were challenged 3 weeks post-inoculation with the phage cocktail toevaluate therapeutic efficacy. Phage cocktail-inoculated grapevines werechallenged at week 3 post-phage inoculation with either XF15 or XF54 toevaluate preventative efficacy of the cocktail. Grapevines from eachtreatment were scored for symptom development twice weekly. To determinedistribution of individual phages comprising the cocktail, grapevineswere assayed at weeks 0, 2, 4, 6, 8, 10, and 12 post-inoculation asdescribed below. Grapevines inoculated in either phage or pathogenchallenge studies were assayed for phage and/or X. fastidiosa infectionat weeks 0, 6, 8, 10, and 12 as described below. Control grapevines wereassayed at 0, 8, and 12 weeks post inoculation to monitor pathogendistribution and disease development. All grapevine assays wereconducted in triplicate with vines containing two cordons. Each cordon(e.g. Cordon1=S1, or Cordon2=S2) was divided into 5-6 (5 inch) segments.Vine segments were numbered from point of inoculation (0) and numberedas below (−) or above (+) the point of inoculation in 5 inch segments.The root portion was divided into three segments: R1, R2, or R3.

Sample Collection and Processing:

For quantification of cocktail phages and pathogens, samples wereobtained as described in Example 13. For assaying of phages, thefiltrate was centrifuged (10,000×g at 4° C. for 15 min) and filtersterilized. The filtrate was used for phage DNA extraction to accomplishquantitative real-time PCR (qRTPCR) (See below). The same protocol wasused for bacterial assays except the pellet was resuspended into 1 ml ofMilli-Q water for isolation of bacterial DNA used in qRTPCR. Average ofqRTPCR results from three segments (e.g. 0a,0b,0c) with similarlocations from triplicate vines was used to determine the CFU and PFU.To determine if phage-resistant X. fastidiosa would develop as a resultof phage challenge experiments, samples collected from grapevines atweek 12 post-pathogen inoculation were processed as described in Example13. Briefly, 100 μl of a suspension of the pellet in 1 ml Milli-Q wasplated on PW-MA (Example 1) supplemented with 40 μg/ml cycloheximide(PW-MAC) and incubated at 28° C. After 10-12 days, individual colonieswere picked and streak-purified 3 times on PW-MAC. Representativeindividual colonies (20 colonies total) from each grapevine sample wereconfirmed at the species and subspecies level using PCR analysis asdescribed by Hernandez-Martinez et al. (Example 1). Phage sensitivity ofconfirmed isolates was determined by the serial dilution spot assay onoverlays and soft agar overlay method as described in Example 3.

PMA Treatment and qRTPCR:

PMA treatment and SYBR-green based qRTPCR protocols were conducted asdescribed in Example 15 using X. fastidiosa-specific primers INF2 (SEQID NO:1) and INR1 (SEQ ID NO:2) and bacteriophage-Xfas303 specificprimers 303-PrimF (SEQ ID NO:5) and 303-PrimR (SEQ ID NO:6), Xfas304specific primers 304-PrimF (SEQ ID NO:3) and 304-PrimR (SEQ ID NO:4),Xfas103-specific primers 103-HelF (SEQ ID NO:7) and 103-HelR (SEQ IDNO:8); and Xfas106-specific primers 106-HelF (SEQ ID NO:9), and 106-HelR(SEQ ID NO:10) listed in Table 5.

TABLE 5 Primers used for qRT_PCR (SEQ ID NOs. 1-10). PrimerSpecific organism Set Sequence and gene Reference INF2GTTTGATTGATGAACGTGGTGAG Xyella fastidosa, Bextine, and INR1CATTGTTTCTTGGTAGGCATCAG gyr B child, 2007 303-PrimF AACTACCTGACAGCGACTXfas303, primase This work 303-PrimR CGTACTAGCTTGGCTTCTA 304-PrimFAAGAAGCGTGGTTTGTTTGC Xfas304, primase This work 304-PrimRCTACCGGCTTCCCTAACTCC 103-HelF AACCTGATCTGGTACGAC Xfas103, helicaseThis work 103-HelR GGACATTTTTCAGTTCTCTC 106-HelF CAACCTCATCTGGTATGACXfas106, helicase This work 106-HelR GTCTTGGGTAATTTCTTTCT *All PCRreactions were conducted for 40 cycles with denaturation at 95° C. for30 sec, annealing at 55° C. for 30 sec and extension at 72° C. for 30sec.

Movement of X. fastidiosa and Disease Development in Grapevines:

Quantitative assessment of duplicate cordons from triplicate samples ofXF15 or XF54 inoculated grapevines showed pathogen distribution ingrapevine segments assayed. qRTPCR detected the presence of an averageof 1×10⁴ and 1×10⁵ CFU/gm of plant tissue (gpt) of XF15 in segment (Seg)S1/1 (cordon 1, 5 inch segment 1 above the point of inoculation) andS2/1 respectively, and an average of 1×10⁴ CFU/gpt of XF54 in S1/2 andS2/2 at week 8-post inoculation. Typical Pierce's Disease symptoms werevisible, such as leaves becoming slightly yellow or red along margins,and leaf margins dried or necrotic by week 8, post-inoculation innon-cocktail challenge grapevines. At week 12 post-inoculation, anaverage of 1×10⁴ and 1×10⁶ CFU/gpt of XF15 was detected in S1/3 andS2/2, respectively. At the same assay interval, an average of 1×10⁵ and1×10⁴ CFU/gpt of XF54 was detected in S1/3 and S2/1, respectively, atweek 12 post inoculation, with grapevines exhibiting PD symptoms. Bothpathogens (XF15 and XF54) were detected in the root system of grapevinesat weeks 8 and 12 post pathogen inoculation at an average of 1×10¹-1×10²CFU/gpt.

Phage Movement and Persistence in Grapevines:

Standard qRTPCR line plots were obtained for phage Xfas303, Xfas304,Xfas103, and Xfas106 that had R2 values of greater than 0.9 andefficiencies of 127%, 123%, 129%, and 120%, respectively. Quantitativeassessment of duplicate cordons from triplicate samples of grapevinesinoculated with phage cocktail (Xfas303, Xfas304, Xfas103, and Xfas106)showed distribution of all phages individually within grapevine segmentsassayed at weeks 2-8 post-cocktail inoculation (FIG. 8). By weeks 8 and12, individual phages were no longer detectable in roots and haddeclined to an average of 1×10¹-1×10² PFU/gpt by week 12 in segmentsassayed with no grapevine symptoms observed (FIG. 8).

Therapeutic Efficacy of Phage Against X. fastidiosa in Grapevines:

Grapevines inoculated with XF15 were challenged with the phage cocktailat three weeks post pathogen inoculation. At 8 weeks (5 weeks postcocktail challenge), the XF15 population was an average of 2-3 logshigher in non-challenged grapevines compared to challenged grapevines.Non-therapeutically treated grapevines showed typical PD symptoms,whereas challenged grapevines did not. At week 12 post-XF15 inoculation(9 weeks post-cocktail challenge), bacterial populations were an averageof 2-3 logs higher in non-challenged grapevines when compared to phagecocktail challenged grapevines (FIG. 9). PD symptoms were not observedin phage challenged grapevines throughout the trial (12 weeks), whereasnon-cocktail treated grapevines exhibited symptoms as early as 4 weeks,which progressed through week 12. Similarly, the bacterial population ingrapevines challenged with XF54-inoculated cocktail declinedsignificantly from weeks 8 through 12 compared to non-challengedgrapevines, with no symptoms observed in cocktail-challenged grapevines.Plating of plant extracts from 12-week cocktail-challenged grapevinesyielded an average of 1×10² CFU/gpt. Representative isolates (20 ea)confirmed as X. fastidiosa from each cordon of each of three grapevineswere all sensitive to the four phages that composed the cocktail.

Prophylactic Efficacy of Cocktail Treatment for the Prevention of PD inGrapevines:

Prophylactic efficacy of the phage cocktail was evaluated by firstinoculating grapevines with the cocktail and then challenging with X.fastidiosa strain XF15 or XF54 at week 3 post-cocktail inoculation.Grapevines treated prophylactically showed no PD symptoms at weeks 8 and12 post-cocktail inoculation. In cocktail-inoculated grapevines thatwere challenged with XF15, pathogen populations reached a maximum of anaverage of 1×10³ CFU/gpt in the segments of the grapevines examined atweeks 8 and 12, and as high as an average of 1×10⁶ CFU/gpt innon-prophylactically treated grapevines. Similar results were observedin grapevines treated prophylactically with cocktail and then challengedwith XF54 at week 3 post phage cocktail inoculation. Plating of plantextracts from 12-week cocktail challenged-grapevines yielded an averageof 3×10² CFU/gpt. Representative isolates (20 ea) confirmed as X.fastidiosa from each cordon from each of three grapevines were allsensitive to the four phages that composed the cocktail.

Persistence and Replication of Phages in Grapevines:

It was of interest to determine phage populations in grapevines in thepresence or absence of introduced hosts (XF15 and XF54). Quantitation ofphage populations in the presence or absence of hosts confirmed that thecocktail phages were able to replicate and maintain higher populationsif sensitive hosts were present in grapevines and then declined in theabsence of a sensitive host in both the therapeutic and prophylacticstudies (FIGS. 10 & 11). Phage populations in non-host containinggrapevines decreased during weeks 8-12, whereas phage populationsincreased an average of 1-2 logs during the same period in grapevinesinoculated with XF15 or XF54 and challenged (therapeutic treatment) withphage cocktail (FIG. 10). Similar results were obtain in prophylacticstudy, with phage populations increasing an average of 1-2 logs overthat observed in non-host containing grapevines (FIG. 11). These resultsconfirmed that bacteriophage treatment prevents or reduces PD symptomsby X. fastidiosa in a plant and demonstrates no adverse effect to atreated plant.

Example 18 Transmission of X. fastidiosa by the Glassy-WingedSharpshooter

The glassy-winged sharpshooter (GWSS), Homalodisca vitripennis, is axylem-feeding leafhopper that transmits X. fastidiosa. The GWSS isprevalent throughout grape growing regions of southern California andTexas. Laboratory-reared X. fastidiosa-free GWSSs were fed on cowpea(Vigna unguiculata subsp. unguiculata) plants harboring either X.fastidiosa or virulent phage Xfas304 for 48 h in three trials to examinethe uptake of X. fastidiosa or phage by GWSS. To determine the abilityof GWSSs to transmit bacteria or phage to plants, GWSSs harboringbacteria or phage were fed on bacteria and phage-free plants. A subsetof bacteria harboring GWSSs were challenged by feeding them on plantsharboring phage for 48 or 96 h. GWSSs and plants were assayedindividually in all experiments to evaluate uptake, transmission orpersistence of bacteria and/or phage using qRTPCR. GWSSs were able touptake and transfer X. fastidiosa and/or phage. In GWSSs harboring X.fastidiosa and challenged with phage, the titer of phage Xfas304increased two-fold, as compared to that observed in X. fastidiosa-freeGWSSs. A two-fold decline in bacterial population was observed in GWSSswhen challenged with phage Xfas304, as compared to non-challenged. GWSSstransmitted X. fastidiosa and/or phage to plants. It is believed thatthis is the first report of phage transfer by GWSSs.

Bacterial Strains, Phages and Inoculum Preparation:

X. fastidiosa strain XF54 (See Example 1) and phage Xfas304 (See Example3) were used in this study. Culture of XF54 was grown on PW-M asdescribed in Example 1. Five-day-old culture of XF54 grown on PW-MA wasused to make bacterial suspensions in phosphate buffer (0.125 M, pH7.1). High-titer phage lysate of Xfas304 (1×10¹⁰ PFU/ml) was preparedand titered as described in Example 3 in sterile deionized water (SDW).

Plant Growth Conditions and Preparation:

Cowpea (Vigna unguiculata subsp. unguiculata) plants were grown in3-inch pots using Metro-Mix and maintained at 24° C. to 29° C. (16 and 8h of light and darkness, respectively) and watered as needed.

Glassy-Winged Sharpshooter:

Laboratory-reared GWSSs were originally reared for multiple generationsin greenhouses at either of two sites: (i) California Department of Foodand Agriculture (CDFA), Arvin or (ii) University of CaliforniaCooperative Extension, San Marcos, Calif. All GWSSs used in this studywere adult males and females and were transported from the above twosites. After receiving, insects were fed on cowpea plants maintained at24° C. to 29° C. (16 and 8 h of light and darkness, respectively), fortwo days to adapt in the new climate before being used in experiments.

Experimental Design:

Each experimental unit (i.e., cage) contained a 15-cm-long stem ofcowpea at the 3-4 leaf stage and a 50 ml flat-bottom tube with a 50-mlsuspension of phage or bacteria in SDW as appropriate. Cowpea stems withattached leaves at the 3-4 leaf stage (cut stem) were collected fromtwo- or three-week-old plants inserted through a hole in the cap andanchored in place with Parafilm (cut stem anchored). GWSSs (3 GWSS/cutstem/cage) were placed in cages and allowed to feed as appropriate.

Uptake of X. fastidiosa and Phage by GWSSs:

To determine uptake of X. fastidiosa and/or phage by GWSSs, cowpea cutstems with attached leaves were anchored in a tube filled with an X.fastidiosa (1×10⁹ CFU/ml) or phage Xfas304 (1×10¹⁰ PFU/ml) suspensionfor 4 h to allow for capillary uptake of X. fastidiosa or phage. Controlcut stems were placed in SDW. After allowing cut stems to uptake theappropriate suspension for 4 h, a subset (3 cut stems) was assayed toquantify X. fastidiosa or Xfas304. After the 4-h uptake period, GWSSs (3GWSSs/cut stem/cage) were allowed to feed on cut stems. Eachexperimental set was done in triplicate (1 cut stem X 3 GWSSs X 3cages). After 48 h, all cowpea cut stems and GWSSs were assayed toquantify the presence of X. fastidiosa and/or phage by qRTPCR. Wateruptake controls were conducted for all experiments under the sameconditions and assayed for X. fastidiosa and phage.

Initial experiments were designed to determine if GWSSs could acquire X.fastidiosa or phage from cut stems that harbored the pathogen or phage,and if so, whether they could transfer the X. fastidiosa or phage toother cut stems. After 48 h, cut stems and GWSSs harbored an average of2×10⁸±1×10⁸ CFU/g of plant tissue (gpt) and 1×10⁶±0.7×10⁶ CFU/GWSS,respectively confirming that GWSSs could acquire X. fastidiosa aspreviously reported (Bextine et al., Biotechniques 38:184, 186, 2005).In a parallel experiment to determine if phage could be acquired byGWSSs from feeding on cut stems, GWSSs assayed after 48 h harbored anaverage of 2×10⁶±0.9×10⁶ PFU/GWSS that was acquired from cut stemscontaining 2×10⁸±1×10⁸ PFU/gpt. The results showed that GWSSs couldacquire phage by feeding on cut stems.

Uptake and Transfer of Phage by GWSSs:

To determine phage uptake and transfer by GWSSs, cowpea cut stems (9)were anchored in 50-ml tubes filled with phage Xfas304 suspension(1×10¹⁰ PFU/ml). Controls (3 cut stems) were placed in SDW. Both sets ofcut stems were allowed to uptake respective medium. After 4 h, three ofthe cut stems allowed to uptake phage were assayed to determine phageconcentration. The remaining 6 cut stems were each placed in individualcages with GWSSs (3 GWSSs/cut stem/cage). After 48 h, 9 GWSSs and theirrespective 3 cut stems were assayed for phage content and the remaining9 GWSSs were transferred to fresh cowpea cut stems anchored in SDW (3GWSSs/cut stem X 3 cages) and allowed to feed for an additional 48 h todetermine phage transfer to cut stems. Cut stems (3) and GWSSs (9) wereassayed for phage after the designated period. Water uptake controlswere conducted for all experiments under the same conditions and assayedfor phage.

Having determined that both phage and bacteria could be acquired byGWSSs, it was of interest to determine if GWSSs that acquired phage fromcut stems could transfer phage and/or bacteria to another cut stem. Asubset of phage-harboring GWSSs were transferred to fresh cowpea cutstems in SDW and allowed to feed. After 48 h, the cut stems and GWSSsharbored an average of 3×10²±2.5×10² PFU/gpt and 3×10³±1.6×10³PFU/GWSSs, respectively, indicating that GWSSs could transfer phage.

Phage Challenge of X. fastidiosa Harboring GWSSs:

To determine if phage could affect the X. fastidiosa population inGWSSs, GWSSs harboring X. fastidiosa were challenged with phage.Briefly, using methods described above with triplicate replicates, GWSSsfed on X. fastidiosa-containing cut stems, verified to contain X.fastidiosa, were transferred to cowpea cut stems uptaking phage Xfas304and allowed to feed. After 48 or 96 h of feeding, the cut stems andGWSSs were assayed for phage and/or X. fastidiosa. For uptake of X.fastidiosa, cowpea cut stems (15) were place in a XF54 suspension (1×10⁹CFU/ml) for 4 h before introducing GWSSs. At 4 h, 3 cut stems wereassayed for X. fastidiosa. Each of the 12 remaining cut stems wereplaced in cages with 3 GWSSs/cut stem and the GWSSs allowed to feed for48 h on the X. fastidiosa-containing cut stems. After 48 h, the X.fastidiosa-fed GWSSs and host cut stems were subdivided into 3 groups:Group 1 was assayed for X. fastidiosa (3 cut stems and 9 GWSSs); Group 2(9 GWSSs) was transferred to fresh cowpea cut stems (3) placed in SDWand allowed to feed for 48 h before GWSSs and cut stems were assayed forX. fastidiosa; Group 3 (18 GWSSs) was transferred to cowpea cut stems(3) placed in a XFas304 suspension (1×10¹⁰ PFU/ml) and allowed to feedfor 48 or 96 h before the GWSSs and cut stems were assayed for X.fastidiosa and phage. Water uptake controls were conducted for allexperiments under the same conditions and assayed for both X. fastidiosaand phage.

36 GWSSs were allowed to feed on cowpea cut stems in a X. fastidiosasuspension and then assayed to determine X. fastidiosa uptake, X.fastidiosa and/or phage transfer, and effect on phage and/or X.fastidiosa in GWSSs. GWSSs (Group 1) allowed to feed on cut stems for 48h that had been placed in a suspension of the X. fastidiosa strain XF54(3×10⁹ CFU/ml) were determined to harbor on the average 1×10⁶±0.7×10⁶CFU/GWSSs and the host feeding cut stems were determined to harbor anaverage of 2×10⁸±1×10⁸ CFU/gpt. After GWSSs harboring X. fastidiosa(Group 2; 1×10⁶±0.7×10⁶ CFU/GWSS) were allowed to feed on fresh cutstems in SDW for 48 h, the cut stems showed an average of 1×10³±1.3×10³CFU/gpt and the GWSSs an average of 2×10³±1×10³ CFU/GWSSs residual X.fastidiosa; reconfirming previous results of X. fastidiosa transfer byGWSSs. Group 3 of the X. fastidiosa harboring GWSSs transferred to cutstems in an Xfas304 suspension (2×10¹⁰ PFU/ml) and allowed to feed for48 h, showed uptake of phage and persistence of X. fastidiosa. Theassayed GWSSs, at 48 h of feeding, harbored an average of 3×10⁴±1.8×10⁴PFU/GWSS of Xfas304 and retained 2×10³±1.1×10³ CFU/GWSSs of XF54. Thecut stems assayed at the same time interval contained an average of3×10⁸±2×10⁸ PFU/gpt and 2×10³±0.6×10³ CFU/gpt. The GWSSs allowed to feedfor 96 h harbored an average of 2×10⁵±1.2×10⁵ PFU/GWSS of Xfas304 and1×10²±0.9×10² CFU/GWSS of XF54, indicating a reduction in XF54 and anaverage 6-fold increase in Xfas304.

Collection and Assay of Cowpea Cut Stems and GWSSs:

GWSSs were sacrificed by freezing at −20° C. for 5 min and cowpea cutstems were collected by cutting at the junction of the tube cap withsterile razor. Each GWSS of each triplicate was placed into 1.5-mlmicro-centrifuge tube with 0.5 ml of P-buffer (50 mM Tris-HCl, pH 7.5,100 mM NaCl, 8 mM MgSO₄), homogenized using a sterile plasticmicro-pestle (Fisher), and filtered through sterile cheesecloth (FisherScientific, USA) to remove tissue debris. Each cut stem of each oftriplicate was weighed and commuted using a sterile razor blade andhomogenized in 1 ml of P-buffer using a mortar and pestle and filteredthrough sterile cheesecloth (Fisher Scientific, USA) to remove tissuedebris. For assaying phage, the filtrate was centrifuged (10,000×g for15 min) and filter sterilized. A portion of filtrate was used for phageDNA extraction as in Example 9, followed by qRTPCR as described below.The remaining portion of the filtrate was used to titer phage asdescribed in Example 3. The same protocol was used for bacterial assays(CFUs), except the pellet was resuspended into 0.5 ml of sterile Milli-Qwater for PMA treatment, bacterial DNA extraction, and qRTPCR asdescribed below.

PMA Treatment and qRTPCR:

PMA treatment and SYBR-green based qRTPCR protocols were conducted asdescribed in Example 17 using X. fastidiosa- and phage-specific primers.

Example 19 Phage Activity Against Xanthomonas axonopodis pv. Citri

Although previous studies have evaluated the use of phage for thecontrol of citrus canker, no conclusive data confirmed the virulentnature of the phages (Balogh et al., Plant Disease, 92:1048-1052, 2008).Only virulent, non-transducing phage should be used to evaluate andimplement a sustainable phage biocontrol system. The sensitivity ofthree Xac field strains (North 40, Block 22, Fort Basinger) obtainedfrom Florida, to two fully characterized virulent phages representativeof the Podoviridae (Xfas303) and the Siphoviridae (Xfas103),respectively, was tested. Results indicate that the three Xac strainstested were only sensitive to phage Xfas303 (FIG. 12). Phage Xfas303 wasable to form clear plaques on three Xac strains tested. 454pyrosequencing was performed of the Xfas303 genome and predicted geneswere fully annotated. The presence of a single subunit RNA Polymerase(SSRNAP) was found, which is indicative of virulent phages such as T7and KMV (Dunn et al., J. Mol. Biol. 166:477-53521, 1983; Lavigne et al.,Virology 312:49-59, 2003). Additionally, it was determined that the typeIV pili of Xanthomonas spp. strain EC-12 is the primary receptor sitefor the phage Xfas303 by making in-frame deletion mutants of pilA in thebacteria. Both phages Xfas303 and Xfas103 adsorb and form clear plaqueson the strain EC-12, but not the ΔpilA derivative, showing results onlyfor plating of phage Xfas303 on EC-12 or EC-12ΔpilA. It was alsodetermined that type IV pili are the primary receptor site for phageXfas303; thus this phage may have a different secondary receptor siterequirement for infection or that phage DNA was restricted. Resultsindicate that the developed non-Xac dependent procedure may be used toisolate virulent phage for Xac with no loss in efficiency of plating(EOP of 0.75).

Example 20 Evidence for Expression of Type IV Pili in Xac

Reports in the literature are conflicting as to the expression and roleof type IV pili in the infection process and pathogenesis of Xac(Brunings et al., Mol. Plant. Pathol. 4:141-57, 2003; Li et al., PLoSONE 6:e21804, 2011; Yang et al., Curr. Microbiol. 48:251-61, 2004). Theresults presented above indicate that all three Xac strains testedexpressed functional type IV pili, as the pili must be retracted tofacilitate adsorption and infection of a phage. Using light microscopystudies the three Xac strains were evaluated for twitching motility, anindicator of functional type IV pili. Pseudomonas aeruginosa strain PAO1and Xanthomonas ssp. strain EC-12 were used as positive controls andEC-12ΔpilA was used as a negative control. The three Xac strains (North40, Block 22 and Fort Basinger) were evaluated for twitching motility.The PAO1, EC-12, and three Xac strains exhibited twitching motility,whereas the EC-12ΔpilA did not. Microscopy studies corroborate resultsobtained with phage sensitivity testing and indicated that three Xacstrain have functional type IV pili that act as an adsorption site forphage Xfas303. Results corroborate the observations of others (Bruningset al., Mol. Plant. Pathol. 4:141-57, 2003; Li et al., PLoS ONE6:e21804, 2011; Yang et al., Curr. Microbiol. 48:251-61, 2004) that typeIV pili are expressed by Xac.

1-34. (canceled)
 35. A plant disease biocontrol composition formulatedfor delivery to a plant, the composition comprising at least onecarrier, and at least one virulent bacteriophage from the Xfas100 phagetype or the Xfas300 phage type, wherein said bacteriophage is virulentto Xylella fastidiosa and Xanthomonas species.
 36. The composition ofclaim 35, wherein the virulent bacteriophage is an active ingredient.37. The composition of claim 36, further defined as being formulated forintroduction to a plant via injection, spraying, misting, or dusting.38. The composition of claim 36, defined as formulated for topicaladministration to a plant.
 39. An isolated bacteriophage that isvirulent to Xylella fastidiosa and Xanthomonas species.
 40. The isolatedbacteriophage of claim 39, further defined as a bacteriophage selectedfrom the group consisting of: Xfas103, Xfas106, Xfas302, Xfas303,Xfas304, and Xfas306 types, wherein representative samples of saidbacteriophages have been deposited under ATCC Accession NumbersPTA-13096, PTA-13095, PTA-13098, PTA-13099, PTA-13100, and PTA-13097.41-46. (canceled)
 47. A method of preventing or reducing symptoms ordisease associated with Xanthomonas axonopodis pv. citri in a plant,comprising contacting said plant with a population of virulentbacteriophage particles that includes Xanthomonas axonopodis pv. citriin its host range.
 48. The method of claim 47, wherein contactingcomprises introducing the bacteriophage particles into the plant. 49.The method of claim 47, wherein the plant is selected from the groupconsisting of a Citrus spp., a Fortunella spp., a Poncirus spp., a lime,a lemon, an orange, a grapefruit, a pomelo, and hybrids of trifoliateorange used for rootstocks.
 50. The method of claim 47, wherein thebacteriophage particles are introduced into the plant by injection, byan insect vector, via the root system, by injection, by spray, by mist,or by dust on the plant.
 51. The method of claim 50, wherein the insectvector is a glassy winged sharpshooter.
 52. The method of claim 47,wherein the number of said bacteriophage to be introduced into saidplant is from 1 to 10¹² PFU/ml.
 53. The method of claim 47, wherein thenumber of said bacteriophage to be introduced into said plant is from10⁴ to 10¹¹ PFU/ml.
 54. The method of claim 47, wherein the number ofsaid bacteriophage to be introduced into said plant is from 10⁷ to 10¹⁰PFU/ml.
 55. The method of claim 47, defined as comprising contacting apopulation of plants with the bacteriophage particles to prevent orreduce symptoms associated with Xanthomonas axonopodis and pathovarsthereof in the population.
 56. The method of claim 47, wherein saidvirulent bacteriophage comprises at least one bacteriophage of a strainselected from the group consisting of: the Xfas100 phage type and theXfas300 phage type.
 57. The method of claim 47, wherein said virulentbacteriophage comprises at least one bacteriophage selected from thegroup consisting of: the Xfas100 phage type and the Xfas300 phage type,wherein the Xfas100 phage type is at least one bacteriophage selectedfrom Xfas103, Xfas106; and the Xfas300 phage type is at least onebacteriophage selected from Xfas302, Xfas303, Xfas304, and Xfas306;wherein representative samples of said phage have been deposited underATCC Accession Numbers PTA-13096, PTA-13095, PTA-13098, PTA-13099,PTA-13100, and PTA-13097, respectively, for phage Xfas103, Xfas106,Xfas302, Xfas303, Xfas304, and Xfas306.
 58. An isolated bacteriophagethat is virulent to Xanthomonas axonopodis further defined asbacteriophage of type Xfas303, wherein a representative sample of saidbacteriophage has been deposited under ATCC Accession Number PTA-13099.